At the age of seventeen Marguerite was married to Charles IV of Alençon, aged twenty, by the decree of King Louis XII (who also arranged the marriage of his ten year old daughter, Claude, to Francis). With this decree, Marguerite was forced to marry a generally kind, but practically-illiterate man for political expediency—"the radiant young princess of the violet-blue eyes... had become the bride of a laggard and a dolt". She had been bartered to save the royal pride of Louis, by keeping the County of Armagnac in the family. There were no offspring from this marriage.

After the death of her first husband in 1525, Marguerite married Henry II of Navarre. Ferdinand II of Aragon had invaded the Kingdom of Navarre in 1512 and Henry ruled only Lower Navarre. Approximately a year after the lead image (in the information box) that was painted by Jean Clouet, on 16 November 1528, Marguerite gave birth to a daughter by Henry, the future Jeanne III of Navarre, who became the mother of the future Henry IV of France.

A Venetian ambassador of that time praised Marguerite as knowing all the secrets of diplomatic art, hence to be treated with deference and circumspection. Marguerite's most remarkable adventure involved freeing her brother, King Francis I, who had been held prisoner in Spain by Charles V, Holy Roman Emperor after being captured in the Battle of Pavia, Italy, 1525. During a critical period of the negotiations, Queen Marguerite rode horseback through wintry woods, twelve hours a day for many days, to meet a safe-conduct deadline, while writing her diplomatic letters at night.

Her only son, Jean, was born in Blois on 7 July 1530, when Marguerite was thirty-eight, an age considered old by sixteenth century standards. The child died on Christmas Day the same year. Scholars believe that her grief motivated Marguerite to write her most controversial work, Miroir de l'âme pécheresse, in 1531.

Sorbonne theologians condemned the work as heresy. A monk said Marguerite should be sewn into a sack and thrown into the Seine. Students at the Collège de Navarre satirized her in a play as "a Fury from Hell". Her brother forced the charges to be dropped, however, and obtained an apology from the Sorbonne.

Following the example set by her mother, Marguerite became the most influential woman in France during her lifetime when her brother acceded to the crown as Francis I in 1515. Her salon, known as the "New Parnassus", became famous internationally.

The writer, Pierre Brantôme, said of her: "She was a great princess. But in addition to all that, she was very kind, gentle, gracious, charitable, a great dispenser of alms and friendly to all."

The Dutch humanist, Erasmus, wrote to her: "For a long time I have cherished all the many excellent gifts that God bestowed upon you; prudence worthy of a philosopher; chastity; moderation; piety; an invincible strength of soul, and a marvelous contempt for all the vanities of this world. Who could keep from admiring, in a great King's sister, such qualities as these, so rare even among the priests and monks?"

Marguerite wrote many poems and plays. She also wrote the classic collection of stories, the Heptameron, as well as a remarkably intense religious poem, Miroir de l'âme pécheresse or Mirror of the Sinful Soul. This particular poem is a first-person, mystical narrative of the soul as a yearning woman calling out to Christ as her father-brother-lover. That her work was passed to the royal court of England provides the basis for conjecture that Marguerite had influence on the Protestant reformation in England.