Wales Under-21 striker Craig Davies, who was only starting because Steve Basham had a stomach upset fired them in front after 20 minutes.
Davies arrived late in the box to prod home Chris Hackett's right-wing cross after good play by Tommy Mooney.
Southend were shell shocked as Oxford then added a second six minutes before half-time. Lucas Cominelli's corner looked to have gone in off the knee of Shrimpers defender Duncan Jupp, but Mooney got the last touch.
Southend had won at Macclesfield in the week to go top, but they struggled to get their game together, despite a lively performance up front from Freddy Eastwood.
And it was Eastwood who gave them a lifeline on the hour when he pounced following a slip by U's keeper Chris Tardif.
Tardif simply missed the ball as it bobbled and Eastwood pounced to slot home from a narrow angle.
However, Southend could not find a way through again as Oxford defended in numbers by getting everyone behind the ball.
Tardif saved well with his legs from Luke Guttridge and in injury-time, he also got behind a 25-yard drive from substitute Wayne Gray.
The outstanding Paul Wanless and Lee Bradbury both nodded headers off the line, but Oxford held on to avenge their 4-0 hiding at Roots Hall last November.