He was born to a noble family in Alexandria. For a short time he taught rhetoric in Pelusium in Egypt; but soon his love for the things of God led him to flee to the Desert as a solitary. After a year of ascetical life, he returned to Pelusium, where he was ordained to the priesthood. After a few years he retired to a monastery where he spent the rest of his life, eventually becoming Abbot. From the monastery he wrote thousands of epistles full of divine grace and wisdom; of these more than two thousand still survive.

Saint Isidore was a student and devout disciple of St John Chrysostom, as he knew him through his writings. When St Cyril became Patriarch of Alexandria, he refused to commemorate St John in the diptychs during the Divine Liturgy. Saint Isidore wrote him a strong letter reminding him not to heed the rumors, prejudices or threats of men, and St Cyril was persuaded to restore commemoration of the Archbishop of Constantinople, and later became a strong advocate of the veneration of St John. Isidore, though a monk, was treated as a spiritual father by Patriarch Cyril: around 433, when St Cyril was inclined to deal harshly with some who had been swept up in the Nestorian heresy, St Isidore wrote to him: 'As your father, since you are pleased to give me this name, or rather as your son, I adjure you to put an end to this dissension lest a permanent breach be made under the pretext of piety.'

With reputation came persecution, and St Isidore suffered much from Imperial and church authorities unhappy with his holy influence. He bore all these troubles impassibly, and in 440 (according to one source) or about 449 (according to another) he joyfully gave up his soul to God.