At Oxford he associated with Robert Sanderson and particularly Robert Boyle, who made Oxford his chief residence from 1654 to 1668. Barlow was a learned Calvinist who opposed Jeremy Taylor and George Bull, and with Thomas Tully was one of the guardians in Interregnum Oxford of acceptable orthodoxy. On the death of John Rouse, Barlow was elected to the librarianship of the Bodleian on 6 April 1652, a post he held until he succeeded to the Lady Margaret professorship in 1660. He favoured the scholars (Anthony à Wood, Anthony Horneck whom he had appointed as chaplain in Queen's, and Thomas Fuller, and was hospitable to Christopher Davenport. He spoke of infant baptism in a letter to John Tombes, which later affected his prospect of preferment.