Mariotto Albertinelli was born in Florence. As a 12-year old boy, he became a pupil of Cosimo Rosselli, and a fellow-pupil with Fra Bartolomeo with whom he formed such an intimate brotherly rapport that in 1494 the two started their own studio in Florence. Vasari's opinion was that Mariotto was not so well grounded in drawing as Bartolomeo, and he tells that, to improve his hand he had taken to drawing the antiquities in the Medici garden, where he was encouraged by Madonna Alfonsina, the mother of Duke Lorenzo II de' Medici.

When the Medici were temporarily banished in 1494, he returned to his friend, whose manner he copied so assiduously, according to Vasari, that his works were taken for Baccio's. When, in the wake of Savonarola's morality campaign, Baccio joined the Dominican order as Fra Bartolomeo in 1500 and gave up painting, Albertinelli, beside himself with the loss, would have joined him; but, spurred by his success in completing an unfinished Last Judgment of Bartolomeo's, he resolved to carry on alone. Among his many students were Jacopo da Pontormo, Innocenzo di Pietro Francucci da Imola and Giuliano Bugiardini.

Mariotto was a most restless person and carnal in the affairs of love and apt to the art of living, and, taking a dislike to the studies and brain-wracking necessary to painting, being also often stung by the tongues of other painters, as is their way, he resolved to give himself to a less laborious and more jovial profession, and so opened the most lovely hostelry outside the Porta San Gallo, and at the sign of the Dragon at the Ponte Vecchio a tavern and inn. This life he led for many months, saying that he had taken up an art that was without muscles, foreshortening or perspective and, better still, without faultfinding, and that the art that he had given up imitated flesh and blood, but this one created flesh and blood; in this if you had good wine you heard yourself praised, but in that every day you were blamed. But at last the low life became an annoyance to him, and, filled with remorse, he returned to painting.

Albertinelli's paintings bear the imprint of Perugino's sense of volumes in space and perspective, Fra Bartolomeo's coloring, the landscape portrayal of Flemish masters like Memling, and Leonardo's Sfumato technique. His chief paintings are in Florence, notably his masterpiece, the Visitation (1503) at the Uffizi.