Born the son of a prosperous London linen draper, Barrow was educated at Cambridge University. Because of his royalist sympathies he was rejected, on Cromwell's instructions, as a candidate for the professorship of Greek. Consequently he began in 1655 an extensive tour of Europe. With the restoration of Charles II, he returned to Britain in 1660 and was finally elected professor of Greek at Cambridge. In 1663 he accepted the newly created Lucasian Professorship of Mathematics, a post he resigned from in 1669.

The claim has often been made that Barrow resigned his chair in favor of his pupil, Isaac Newton. In reality Newton was not the pupil of Barrow and, while he appreciated Newton's mathematical genius and saw to it that Newton succeeded him, Barrow was more interested in advancing his own career. He was appointed chaplain to Charles II in 1669 and in 1673 returned to Cambridge as Master of Trinity College, an office in the gift of the king. He died soon after in 1677 from, according to John Aubrey, an overdose of opium, an addiction that he had acquired in Turkey.

Barrow is best known for his Lectiones opticae (1669) and Lectiones geometricae (1670), both edited by Newton, and the posthumously published Lectiones mathematicae (1683). Unfortunately for Barrow's reputation, his work in both optics and mathematics was soon overshadowed by Newton's own publications.