On the Restoration, Barlow was one of the commissioners for restoring the members of the university who had been ejected in 1648 and expelling the intruders. On behalf of John Owen, molested for preaching in his own house, he mediated with Edward Hyde, the Lord Chancellor. Henry Wilkinson was removed as Lady Margaret Professor of Divinity on 25 September 1660, in favour of Barlow. A few days before, on 1 September, he had taken his degree of D.D., one of a batch of loyalists created doctors by royal mandate. On the death of Barton Holiday in 1661, Barlow was appointed Archdeacon of Oxford, but there was a delay caused by a dispute between him and Thomas Lamplugh, ultimately decided in Barlow's favour, who was eventually installed on 13 June 1664. Barlow meanwhile was accused by Wood of underhand meddling in the election of Thomas Clayton to the wardenship of Merton College in 1661. Barlow wrote at the request of Robert Boyle an elaborate treatise on "Toleration in Matters of Religion" at this time, but it was not published until after his death (in Cases of Conscience, 1692). Barlow's reasoning is based more on expediency than on principle. He shows that the religious toleration he advocates does not extend to atheists, papists or Quakers. Earlier, when Jews were applying to Cromwell for readmission into England, Barlow had composed "at the request of a person of quality" a tract on "Toleration of the Jews in a Christian State", published in the same collection. On the other hand, Barlow was one of a group of Oxford grandees hostile to the Royal Society, along with John Fell, Obadiah Walker, and Thomas Pierce. He was an enemy of the "new philosophy" (as propounded by leading Royal Society members), giving as his confessional reasons that it was "impious if not plainly atheistic, set on foot and carried on by the arts of Rome," so designing to ruin the Protestant faith by disabling men from defending the truth. He noted the Catholic background of Descartes, Gassendi, Mersenne and Du Hamel. His Directions to a young Divine for his Study of Divinity of this period contain a catalogue of theological works classified by subjects, with remarks on their value and character. As pro-vice-chancellor of the university in 1673, he called in question William Richards, Chaplain of All Souls College, for Arminian doctrine in a sermon at St Mary's.