And it was a brilliant effort from the 35-year-old left-back that settled this battle between the top two in League Two.
Breaking into the box, Humphreys met Eoin Doyle's left-wing cross with a classy volley as he guided the ball into the far corner from 10 yards.
It was touch and go whether Humphreys would play, but Chesterfield boss Paul Cook will be glad he did as Chesterfield - missing top scorer Marc Richards - maintained their best-ever start to a season.
Oxford had skipper Andy Whing sent off after 67 minutes for a reckless challenge on Doyle as they surrendered their unbeaten record.
Chesterfield might have added further goals with U's keeper Ryan Clarke stopping a near-post effort from Tendayi Darikwa and turning aside Jay O'Shea's drive.
Deane Smalley drove wide from a good position and also headed against a post late on, but once Whing was dismissed, Oxford were always up against it as Cook's team chalked up a sixth clean sheet in eight games.