Rosso received his early training in the studio of Andrea del Sarto, alongside his contemporary Jacopo da Pontormo (Pontormo, Jacopo da). The earliest works of these two young painters combined influences from Michelangelo and from northern Gothic engravings in a novel style, which departed from the tenets of High Renaissance art and was characterized by its highly charged emotionalism and departure from classicism. Rosso's most remarkable paintings from this period are the Assumption (1517), the Deposition (1521), and Moses Defending the Daughters of Jethro (c. 1523).

At the end of 1523 Rosso moved to Rome, where his exposure to Michelangelo's Sistine ceiling, the late art of Raphael, and the work of Parmigianino resulted in a radical realignment of his style. His Dead Christ with Angels (c. 1526) exemplifies this new style with its feeling for rarefied beauty and subdued emotion. Fleeing from the sack of the city in 1527, he worked briefly in several central Italian towns. In 1530, on the invitation of Francis I, he went to France (by way of Venice) and remained in the royal service there until his death.

Rosso's principal surviving work is the decoration of the Galerie François I at the palace of Fontainebleau (c. 1534–37), where, in collaboration with Francesco Primaticcio (Primaticcio, Francesco), he developed an ornamental style whose influence was felt throughout northern Europe. His numerous designs for engravings also exercised a wide influence on the decorative arts both in Italy and in northern Europe.