Having also been defeated against Crawley, progression to the knock-out stages is now impossible with a game still to play.
Southampton went ahead when Sam McQueen headed home a near post corner with only eight minutes on the clock.
Young winger Dion Sembie-Ferris got his first ever U's goal on twenty two minutes to level things up but it wasn't enough for a young U's side.
Olufela Olomola smashed home the winner after the break, keeping the Saints hopes of progression out of the group alive.
Having had the better of the early possession, the visiting side were denied on four minutes by good defending by Luke Prosser to prevent Cook getting a touch at the far post.
That warning from a set piece wasn’t heeded four minutes later though, as Sam McQueen headed home at the near post from a Saints corner.
James Bransgrove then made a low save to prevent the Saints extending the lead, diving to his left to keep out O’Connor.
It took fifteen minutes for the U’s to test Lewis in the away goal, and it was a comfortable save from a Denny Johnstone header.
Craig Slater then tried his luck from just outside the area, but shot over the bar after a clever one-two got him space twenty yards out.
That came on twenty one minutes and seconds later, the U’s had levelled the scores.
Sammie Szmodics broke through, rode a challenge from a Saints centre half and rolled a perfect ball into Dion Sembie-Ferris’ path.
The young winger didn’t have to break stride or take a touch, he hit a sweet left foot shot across the keeper and into the bottom corner to make it 1-1.
Glen Kamara and Szmodics then combined, allowing the latter to shoot - a defender getting a touch to deflect it for a corner.
Saints had a couple of openings as the half wore on, with Johnson lobbing wide after a clearance from Bransgrove and Olomola’s shot not finding the target either.
The U’s were unfortunate not to take the lead on forty two minutes, with an extremely harsh decision to disallow what looked a perfectly good goal.
Under pressure from Johnstone, Saints keeper Lewis dropped the ball behind him and the U’s striker sidefooted home into the empty net.
No appeals came from the visiting side but the referee blew for a foul and the U’s celebrations were short-lived.
Goalscorer Sembie-Ferris had taken a heavy knock to his thigh towards the half time break and was the youngster was unable to continue after the interval.
An even younger winger replaced him, with Tariq Issa coming on the left wing having scored the winner for the U’s U23s at Hull just two days previously.
Five minutes after the break, the U’s found themselves behind again, as Olomola struck for the visitors.
Saints captain Harrison Reed drove down the right, beat Lewis Kinsella and pulled the ball back for Olomola to lash home high into the net.
Just after the hour mark, Chris Porter replaced Johnstone and on seventy three minutes, Cameron James was introduced for Drey Wright.
A flowing move from the Saints, again instigated by Reed, ended with Johnson being denied by a great low save from Bransgrove.
Two of the U’s subs combined on seventy five minutes, with James finding Issa who shot caught a defender before ending up in Lewis’ hands.
Fellow sub Porter then created another chance for Issa, with a knock down that still left a lot to be done.
Issa showed some sublime skill to give himself space to shoot and was denied by a smart save to his left from Lewis.
That was the last chance the U’s had and the final whistle went with the Saints running out 2-1 winners in what was an entertaining match between two young footballing sides.
It also meant that the U’s couldn’t progress into the knock-out stages of the competition, with a home game against Charlton Athletic still to come.