He was the son of Ercole I d'Este and Eleanor of Naples and became duke on Ercole's death in June 1505.
In the first year of his rule he uncovered a plot by his brother Ferrante and half-brother Giulio d'Este, directed against him and his other brother Ippolito. In September 1506 a trial for lèse majesté and high treason was held and, as expected, the death sentence was passed, but just as Ferrante and Giulio were about to mount the gallows they were informed that the duke had commuted their sentence to life imprisonment. They were led away to two cells in the Torre dei Leoni. Ferrante died in his cell after 34 years of imprisonment, while Giulio held on until he was pardoned in 1559, after 53 years of imprisonment. After his release, Giulio was ridiculed in the streets of Ferrara for his outdated clothes and died in 1561.
In the Italian Wars Alfonso preserved his precarious position among the contending powers by flexibility and vigilance and the unrivalled fortifications of Ferrara; he entered the League of Cambrai against Venice and remained an ally of Louis XII of France even after Pope Julius II had made peace with Venice; when the Bolognesi rebelled against Julius and toppled Michelangelo's bronze statue of the Pope from above the gate, Alfonso received the shards and recast them as a cannon named La Giulia, which he set on the ramparts of the castello: in 1510 Julius excommunicated him and declared his fiefs forfeit, thereby adding Ferrara to the Papal States; Alfonso then fought successfully against the Venetian and Papal armies, gaining the Battle of Polesella, capturing Bologna, and playing a major part in the French victory at the Battle of Ravenna (1512). These successes were based on Ferrara's artillery, produced in his own foundry which was the best of its time. In both of his portraits by Titian, (Compare illustration above) he poses with his arm across the mouth of one of his cannon.
In 1526–1527 Alfonso participated in the expedition of Charles V, Holy Roman Emperor and king of Spain, against Pope Clement VII, and in 1530 the pope again recognized him as possessor of the forfeited duchies of Modena and Reggio.
Alfonso's first wife was Anna Sforza, the sister of Gian Galeazzo Sforza. His second wife was Lucrezia Borgia.