Oxford had the first word but Chesterfield had the last as Chris Atkinson's stoppage-time goal gave the home side a 2-1 victory after going behind in the opening minute.
John Mullins headed Oxford ahead but Liam Cooper equalised on the stroke of half-time and Atkinson settled it right at the end.
Tommy Lee returned to the side after two months out through injury but as beaten in the first minute when a corner to the back post was headed just under the bar by Mullins.
Lee kept Chesterfield in it with a flying save to parry a Jake Forster-Caskey header six minutes later, but Oxford should have scored again in the 33rd minute when a cross fell invitingly to James Constable 10 yards out and he lashed a shot high over the bar.
Chesterfield had created few chances but they equalised in first-half stoppage time when a corner broke to Cooper, who fired into the roof of the net from 12 yards.
It needed a brave block by Andy Whing to stop Brennan Dickenson putting the home side ahead in the 66th minute but Chesterfield won it in added time when Marc Richards put Atkinson in to slide a low shot across Ryan Clarke and inside the far post.