Leonardo da Vinci had begun working on a portrait of Lisa del Giocondo, the model of the Mona Lisa, by October 1503. It is believed by some that the Mona Lisa was begun in 1503 or 1504 in Florence. Although the Louvre states that it was "doubtless painted between 1503 and 1506", art historian Martin Kemp says that there are some difficulties in confirming the dates with certainty.

Circa 1505, Raphael executed a pen-and-ink sketch, in which the columns flanking the subject are more apparent. Experts universally agree that it is based on Leonardo's portrait. Other later copies of the Mona Lisa, such as those in the National Museum of Art, Architecture and Design and The Walters Art Museum, also display large flanking columns. As a result, it was thought that the Mona Lisa had been trimmed.

The Mona Lisa began influencing contemporary Florentine painting even before its completion. Raphael, who had been to Leonardo's workshop several times, promptly used elements of the portrait's composition and format in several of his works, such as Young Woman with Unicorn (c. 1506), and Portrait of Maddalena Doni (c. 1506). Later paintings by Raphael, such as La velata (1515–16) and Portrait of Baldassare Castiglione (c. 1514–15), continued to borrow from Leonardo's painting.

In 1516, Leonardo was invited by King Francis I to work at the Clos Lucé near the Château d'Amboise; it is believed that he took the Mona Lisa with him and continued to work on it after he moved to France.

The record of an October 1517 visit by Louis d'Aragon states that the Mona Lisa was executed for the deceased Giuliano de' Medici, Leonardo's steward at the Belvedere Palace between 1513 and 1516—but this was likely an error. According to Vasari, the painting was created for the model's husband, Francesco del Giocondo. A number of experts have argued that Leonardo made two versions (because of the uncertainty concerning its dating and commissioner, as well as its fate following Leonardo's death in 1519, and the difference of details in Raphael's sketch—which may be explained by the possibility that he made the sketch from memory). The hypothetical first portrait, displaying prominent columns, would have been commissioned by Giocondo circa 1503, and left unfinished in Leonardo's pupil and assistant Salaì's possession until his death in 1524. The second, commissioned by Giuliano de' Medici circa 1513, would have been sold by Salaì to Francis I in 1518 and is the one in the Louvre today. Other believe that there was only one true Mona Lisa, but are divided as to the two aforementioned fates. The famed painting was kept at the Palace of Fontainebleau, where it was kept until Louis XIV moved it to the Palace of Versailles, where it remained until the French Revolution. In 1797, it went on permanent display at the Louvre.

A version of the Mona Lisa known as the Isleworth Mona Lisa and also known as the Earlier Mona Lisa was first bought by an English nobleman in 1778 and was rediscovered in 1913 by Hugh Blaker, an art connoisseur. The painting was presented to the media in 2012 by the Mona Lisa Foundation. It is a painting of the same subject as Leonardo da Vinci's Mona Lisa. The painting is claimed by a majority of experts to be mostly an original work of Leonardo dating from the early 16th century. Other experts, including Zöllner and Kemp, deny the attribution.

The first and most extensive recorded cleaning, revarnishing, and touch-up of the Mona Lisa was an 1809 wash and revarnishing undertaken by Jean-Marie Hooghstoel, who was responsible for restoration of paintings for the galleries of the Musée Napoléon. The work involved cleaning with spirits, touch-up of colour, and revarnishing the painting.

After the French Revolution, the painting was moved to the Louvre, but spent a brief period in the bedroom of Napoleon (d. 1821) in the Tuileries Palace. The Mona Lisa was not widely known outside the art world, but in the 1860s, a portion of the French intelligentsia began to hail it as a masterwork of Renaissance painting.

During the Franco-Prussian War (1870–1871), the painting was moved from the Louvre to the Brest Arsenal.

By the early 20th century, some critics started to feel the painting had become a repository for subjective exegeses and theories. Upon the painting's theft in 1911, Renaissance historian Bernard Berenson admitted that it had "simply become an incubus, and he was glad to be rid of her." Jean Metzinger's Le goûter (Tea Time) was exhibited at the 1911 Salon d'Automne and was sarcastically described as "la Joconde à la cuiller" (Mona Lisa with a spoon) by art critic Louis Vauxcelles on the front page of Gil Blas. André Salmon subsequently described the painting as "The Mona Lisa of Cubism".

In 1906, Louvre restorer Eugène Denizard performed watercolour retouches on areas of the paint layer disturbed by the crack in the panel. Denizard also retouched the edges of the picture with varnish, to mask areas that had been covered initially by an older frame.

The Mona Lisa has had many different decorative frames in its history, owing to changes in taste over the centuries. In 1909, the art collector Comtesse de Béhague gave the portrait its current frame, a Renaissance-era work consistent with the historical period of the Mona Lisa.

By 1911, the painting was still not popular among the lay-public.

On 21 August 1911, the painting was stolen from the Louvre.

Peruggia was an Italian patriot who believed that Leonardo's painting should have been returned to an Italian museum. Peruggia may have been motivated by an associate whose copies of the original would significantly rise in value after the painting's theft. After having kept the Mona Lisa in his apartment for two years, Peruggia grew impatient and was caught when he attempted to sell it to Giovanni Poggi, director of the Uffizi Gallery in Florence. It was exhibited in the Uffizi Gallery for over two weeks and returned to the Louvre on 4 January 1914.

The Mona Lisa has survived for more than 500 years, and an international commission convened in 1952 noted that "the picture is in a remarkable state of preservation." It has never been fully restored, so the current condition is partly due to a variety of conservation treatments the painting has undergone. A detailed analysis in 1933 by Madame de Gironde revealed that earlier restorers had "acted with a great deal of restraint."

Salvador Dalí, famous for his surrealist work, painted Self portrait as Mona Lisa in 1954.

On 30 December 1956, Bolivian Ugo Ungaza Villegas threw a rock at the Mona Lisa while it was on display at the Louvre. He did so with such force that it shattered the glass case and dislodged a speck of pigment near the left elbow. The painting was protected by glass because a few years earlier a man who claimed to be in love with the painting had cut it with a razor blade and tried to steal it.

In the early 21st century, French scientist Pascal Cotte hypothesized a hidden portrait underneath the surface of the painting, circumstantial evidence for which was produced using reflective light technology.

Before the 1962–1963 tour, the painting was assessed for insurance at $100 million (equivalent to $650 million in 2018), making it, in practice, the most highly-valued painting in the world. The insurance was not purchased; instead, more was spent on security.

From December 1962 to March 1963, the French government lent it to the United States to be displayed in New York City and Washington, D.C. It was shipped on the new ocean liner SS France.

Andy Warhol created serigraph prints of multiple Mona Lisas, called Thirty Are Better than One, following the painting's visit to the United States in 1963.

In 1974, the painting was exhibited in Tokyo and Moscow.

On 21 April 1974, while the painting was on display at the Tokyo National Museum, a woman sprayed it with red paint as a protest against that museum's failure to provide access for disabled people.

In 1977, a new insect infestation was discovered in the back of the panel as a result of crosspieces installed to keep the painting from warping. This was treated on the spot with carbon tetrachloride, and later with an ethylene oxide treatment.

In recent decades, the painting has been temporarily moved to accommodate renovations to the Louvre on three occasions: between 1992 and 1995, from 2001 to 2005, and again in 2019.

Research in 2003 by Professor Margaret Livingstone of Harvard University said that Mona Lisa's smile disappears when observed with direct vision, known as foveal. Because of the way the human eye processes visual information, it is less suited to pick up shadows directly; however, peripheral vision can pick up shadows well.

On 6 April 2005—following a period of curatorial maintenance, recording, and analysis—the painting was moved to a new location within the museum's Salle des États. It is displayed in a purpose-built, climate-controlled enclosure behind bulletproof glass.

Research in 2008 by a geomorphology professor at Urbino University and an artist-photographer revealed likenesses of Mona Lisa's landscapes to some views in the Montefeltro region in the Italian provinces of Pesaro and Urbino, and Rimini.

On 2 August 2009, a Russian woman, distraught over being denied French citizenship, threw a ceramic teacup purchased at the Louvre; the vessel shattered against the glass enclosure.

A version of Mona Lisa known as Mujer de mano de Leonardo Abince ("Woman by Leonardo da Vinci's hand") held in Madrid's Museo del Prado was for centuries considered to be a work by Leonardo. However, since its restoration in 2012, it is considered to have been executed by one of Leonardo's pupils in his studio at the same time as Mona Lisa was being painted. The Prado's conclusion that the painting is probably by Salaì (1480–1524) or by Melzi (1493–1572) has been called into question by others.

In 2014, 9.3 million people visited the Louvre. Former director Henri Loyrette reckoned that "80 percent of the people only want to see the Mona Lisa."

In 2014, a France 24 article suggested that the painting could be sold to help ease the national debt, although it was noted that the Mona Lisa and other such art works were prohibited from being sold due to French heritage law, which states that "Collections held in museums that belong to public bodies are considered public property and cannot be otherwise."

A new queuing system introduced in 2019 reduces the amount of time museum visitors have to wait in line to see the painting. After going through the queue, a group has about 30 seconds to see the painting.

On the 500th anniversary of the master's death, the Louvre held the largest ever single exhibit of Da Vinci works, from 24 October 2019 to 24 February 2020. The Mona Lisa was not included because it is in such great demand among visitors to the museum; the painting remained on display in its gallery.