His uncle Guidobaldo I of Urbino, who was heirless, called him at his court, and named him as heir of that dukedom in 1504 through the intercession of Julius II. In 1502 the della Rovere had lost the seigniory of Senigallia, occupied by Cesare Borgia, then the most powerful figure in the Marche: Francesco Maria and his mother were saved from the slaughter perpetrated by Borgia's troops by the then-land soldier Andrea Doria. When in 1508 Guidobaldo died, Francesco Maria became duke of Urbino; thanks to the support of his uncle Giuliano della Rovere, now pope as Julius II, he could also recover Senigallia after Borgia's death.
In 1509 he was appointed as capitano generale (commander-in-chief) of the Papal States, and subsequently fought in the Italian Wars against Ferrara and Venice. In 1511, after he had failed to conquer Bologna, he had the cardinal killed by his troops, a cruel action for which he was compared to Borgia himself. In 1513 he was created also lord of Pesaro.
However, the death of Julius II deprived him of his main political patron, and under the new pope, Leo X Medici, Pesaro was given to the latter's nephew, Lorenzo II de' Medici. In 1516 he was excommunicated and ousted from Urbino, which he tried unsuccessfully to recover the following year (see War of Urbino). He could return in his duchy only after Leo's death (1521).
Della Rovere fought as capitano generale of the Republic of Venice in Lombardy during the Italian Wars of 1521 (1523–1525), but with the new Medici Pope, Clement VII, the della Rovere were increasingly marginalized. As supreme commander of the Holy League, his inaction against the Imperial invasion troops is generally listed as one of the cause of the Sack of Rome (1527).
He was a protagonist of the capture of Pavia in the late 1520s, and later fought for the Republic of Venice. Later he arranged the marriage of son Guidobaldo to Giulia da Varano (belonging to another former seigniory family of the region) to counter the Papal power in the Marche.
He died in Pesaro, poisoned. Some scholars suggest that The Murder of Gonzago, an unknown play referenced in William Shakespeare's Hamlet, Prince of Denmark, which is itself later reworked by Hamlet into The Mousetrap (the play within the play), may have been a popular theatrical reenactment of Della Rovere's death and may have been portrayed in England's early theaters during the Elizabethan Era.