He was born in Siena, the nephew of Aeneas Silvius Piccolomini, the future Pope Pius II, by his sister Laodamia. He was received as a boy into the household of Aeneas Silvius, who permitted him to assume the name and arms of the Piccolomini family (his brother Antonio being made Duke of Amalfi during the pontificate of Pius II). Pius II appointed him in 1460, when only 22 years of age, to the see of Siena, which he had just raised to an archbishopric and made him a cardinal, at his first consistory, 5 March 1460. Within months he sent him as legate to the March of Ancona, with the experienced bishop of Marsico as his counsellor. He proved studious and effective.
Cardinal Piccolomini participated in the conclave that elected Pope Paul II (1464–71) in 1464 but was absent when Pope Sixtus IV (1471–84) was elected in 1471. He was employed in several important legations, as by Paul II at the Imperial diet at Regensburg/Ratisbon, and by Sixtus IV to secure the restoration of ecclesiastical authority in Umbria. He participated in the conclave of 1484 which elected Innocent VIII and in the conclave of 1492 which elected Alexander VI. The cardinal was involved in Alexander's brief-lived effort to reform the Roman curia, following the murder of his son Giovanni Borgia (1474) in 1497.
In 1502 the Cardinal commissioned a library with access from an aisle of the Duomo di Siena, which was intended to house the library of humanist texts assembled by his uncle, and commissioned the artist Pinturicchio to fresco its vault and ten narrative panels along the walls depicting scenes from the life of Aeneas Silvius Piccolomini. Though Pinturrichio labored for five years, in the event, the books never reached their splendid destination; yet the Piccolomini Library is a monument of the High Renaissance in Siena. It gives an edited version of Pius' life, passing over his former support of the antipope Felix V.
Amid the disturbances consequent upon the death of the Borgia Pope Alexander VI (1492–1503), it took the combined pressures of all the ambassadors to induce Cesare Borgia to withdraw from Rome, so that an unpressured conclave might take place. In it, Cardinal Piccolomini was elected Pope Pius III on September 22, 1503. This selection can be seen as a compromise between factions, Borgia and della Rovere, picking a frail cardinal with long experience in the Curia over the kin of either Sixtus IV or Alexander VI. His coronation took place on October 8, 1503. He at once took in hand the reform of the papal court and arrested Cesare Borgia; but after a brief pontificate of twenty-six days he died (October 18, 1503) of an ulcer in the leg, or, as some have alleged, of poison administered at the instigation of Pandolfo Petrucci, governor of Siena.