Elliot Lee grabbed the opening goal of the game midway through the first-half, as the Luton loanee calmly slotted home past Joe Day to put Oxford ahead.
Rovers had the majority of first half chances, with both James Daly and George Williams putting Jack Stevens to work in the Oxford goal.
Oxford added a second goal late on in stoppage time, as Barker found the net from inside the Rovers area.
Rovers made three changes from the side that faced Fleetwood on Saturday.
The trip to Highbury saw Rovers pick up a sixth clean sheet of the season, with the entire back five remaining unchanged for the visit of Karl Robinson’s Oxford.
The trio of changes came in midfield and attack – as Oztumer, McCormick and Rodman came into the fold. Hanlan and Ayunga gave Paul Tisdale attacking options on the bench, with Sam Nicholson also named amongst the substitutes.
Oxford’s prolonged winning streak was ended on Saturday in Yorkshire by Doncaster Rovers, but United didn’t surrender their goalscoring form at the Keepmoat, with Oxford scoring at least two goals in seven of their last eight matches heading into Tuesday night.
The first opening of the game fell to the away side, as a cross to the far post swung into the path of James Henry. The winger couldn’t shake off the attention from Jack Baldwin to make a connection with the cross and the ball sailed out of play.
Rovers had to endure the mainstay of the defending of the in the early exchanges of the first half – but limited the clear cut chances for the home side.
As the first-half progressed Rovers grew into the contest and created the best chance of the half just after the twenty-minute mark. An inviting cross from George Williams from wide out of the right picked out James Daly inside the Oxford area. The striker has managed to navigate a run to split the Oxford defenders, but his first-time effort was hit low and have the direction to test Jack Stevens.
Daly’s chance started a bright spell for the Gas, as Luke McCormick quickly followed the effort up with a set-piece that climbed over the target, but showed good attacking intent from 25-yards-out to for goal.
The Rovers front three looked energetic and looked to adapt play down both flanks. A ball down the channel from McCormick was chased down by James Daly, with the striker snatching possessions on the edge of the Oxford area. A neat touch saw the striker advance closer to goal, but a from a tight angle, he could only direct his effort high and wide of goal.
Rovers were made to rue not scoring through their spell of chances as Elliot Lee broke the deadlock in the 29th minute. A flick forward from James Henry set Lee on a break down the right. The striker managed to dart inside the Rovers area before drawing in Joe Day and placing a finish beyond the Rovers ‘keeper to give Oxford the lead.
The response from Rovers from conceding the goal was positive. Oztumer managed to jinx into the Oxford area to aim shot across goal that almost fell favourably for both Rodman and Daly, who both met inside the six-yard area to attack any rebounds on offer.
Soon after, George Williams showed good feet to twist and turn inside the visitor’s penalty area and force a strong save from Stevens, as the Oxford ‘keeper tipped Williams’ forceful effort over the crossbar.
From the resulting corner, Luke Leahy managed to attack the delivery, as the defender guided a powerful header the ruffled the side netting.
Rovers finished the first-half strong, escalating their half-chances on goal and enjoying the majority of the attacking play, but were unable to dent the away side’s slim advantage at the interval.
Paul Tisdale’s side carried on in the same fashion after the break. Alex Rodman took sight on goal from 20-yards out and forced Stevens into making a routine stop.
Rovers enjoyed plenty of the ball in and around the Oxford half. Josh Grant was able to glide a useful cross towards the far-post where George Williams was denied a chance to glance home a close-range finish as Atkinson headed the delivery behind for a corner.
As the hour mark passed, Rovers turned to the bench, introducing both Westbrooke and Nicholson.
The changes almost had an instant impact, as Nicholson was on hand to pick up a slack clearance from the away side and showed a good turn of pace to break towards goal. The winger cut inside to central position, before his low effort was held by Stevens.
Rovers initiated more attacking options from the bench, as Brandon Hanlan replaced Alex Rodman with twenty minutes left on the clock.
Oxford were able to manage the game through a series of substitutions and succeeded to slow the pace of the encounter as the encroached into the final ten minutes.
There were late signs of danger as substitute Skyes broke through on goal , but a last ditch tackle from Luke Leahy proved to be enough to steer the striker’s effort over the target.
Joe Day was called upon moments later, as the goalkeeper produced fine one-handed save to tip wide a header from Winnall that looked destined for the top corner.
Rovers were looking for a final chance and that came when a cross from the right was flicked towards the far-post by Ayunga. The delivery picked out both McCormick and Nicholson unmarked, with the former drilling a volley inches wide of the target.
In stoppage time, Oxford added the security of a second goal, with substitute Barker firing home from inside the area.
Rovers continue a run of home games on Saturday, as they entertain not so distant neighbours, Swindon Town.
Rovers: Joe Day, Luke Leahy, Alfie Kilgour (63’ Zain Westbrooke), Max Ehmer (C), Jack Baldwin, George Williams, Luke McCormick, Josh Grant, Alex Rodman (70’ Brandon Hanlan, Erhun Otzumer (63’ Sam Nicholson), James Daly (80’ Jonah Ayunga).
Subs: Jordi Van Stappershoef, Cian Harries, Cameron Hargreaves, Jonah Ayunga, Brandon Hanlan.
Oxford United: Jack Stevens, Josh Ruffels, Elliott Moore (c), Alex Gorrin, Cameron Brannagan, Matty Taylor (75’ Mark Sykes), Sam Long (39’ Anthony Forde), James Henry (58’ Brandon Barker), Rob Atkinson, Olamide Shodipo (58’ Sam Winnall), Elliott Lee (75’ Liam Kelly).
Subs: Simon Eastwood, Joe Grayson