Agapetus was born in Rome, although his exact date of birth is unknown. He was the son of Gordianus, a Roman priest who had been slain during the riots in the days of Pope Symmachus (term 498–514). The name of his father might point to a familial relation with two other Popes: Felix III (483–492) and Gregory I (590–604). Gregory was a descendant of Felix. Gregory's father, Gordianus, held the position of Regionarius in the Roman Church. Nothing further is known about the position.
Agapetus collaborated with Cassiodorus in founding at Rome a library of ecclesiastical authors in Greek and Latin and helped Cassiodorus with the project of translating the standard Greek philosophers into Latin.
Jeffrey Richards describes him as "the last survivor of the Symmachan old guard", having been ordained as a deacon perhaps as early as 502, during the Laurentian schism. He was elevated from archdeacon to pope in 535. His first official act was to burn, in the presence of the assembled clergy, the anathema which Boniface II had pronounced against the latter's deceased rival Dioscurus on a false charge of simony and had ordered to be preserved in the Roman archives.
He confirmed the decrees of the Council of Carthage, after the retaking of North Africa from the Vandals, according to which converts from Arianism were declared ineligible to Holy Orders and those already ordained were merely admitted to lay communion. He accepted an appeal from Contumeliosus, Bishop of Riez, whom a council at Marseilles had condemned for immorality, and he ordered Caesarius of Arles to grant the accused a new trial before papal delegates.
Meanwhile, the Byzantine general Belisarius was preparing for an invasion of Italy. King Theodahad of the Ostrogoths begged Agapetus to proceed on an embassy to Constantinople and use his personal influence to appease Emperor Justinian I following the death of Amalasuntha. To defray the costs of the embassy, Agapetus pledged the sacred vessels of the Church of Rome. He set out in mid-winter with five bishops and a large retinue. In February 536, he appeared in the capital of the East. Agapetus immediately turned his attention from the political matter Theodahad had sent him to address to a religious one.
The occupant of the Byzantine patriarchal see was Anthimus I, who had left his episcopal see of Trebizond. Against the protests of the orthodox, the Empress Theodora finally seated Anthimus in the patriarchal chair. When Agapetus arrived members of the clergy entered charges against Anthimus as an intruder and a heretic. Agapetus ordered him to make a written profession of faith and to return to his forsaken see; upon Anthimus' refusal, Agapetus deposed him. The Emperor threatened Agapetus with banishment. Agapetus is said to have replied, "With eager longing have I come to gaze upon the Most Christian Emperor Justinian. In his place I find a Diocletian, whose threats, however, terrify me not." Agapetus, for the first time in the history of the Church, personally consecrated Anthimus' legally elected successor, Mennas. Justinian delivered to the Pope a written confession of faith, which the latter accepted with the proviso that "although he could not admit in a layman the right of teaching religion, yet he observed with pleasure that the zeal of the Emperor was in perfect accord with the decisions of the Fathers". Four of Agapetus' letters have survived. Two are addressed to Justinian in reply to a letter from the emperor, in the latter of which he refuses to acknowledge the Orders of the Arians. A third is addressed to the bishops of Africa, on the same subject. The fourth is a response to Reparatus, Bishop of Carthage, who had sent him congratulations upon his elevation to the Pontificate.
Shortly afterwards, Agapetus fell ill and died on 22 April 536, after a reign of just ten months. His remains were brought in a lead coffin to Rome and deposited in St. Peter's Basilica.
Agapetus I has been canonised by both the Roman Catholic and Orthodox traditions. His memory is kept on 20 September, the day of his deposition, in the Roman Catholic Church. The Eastern churches commemorate him on 22 April, the day of his death.