Barnsley started with a bang, scoring through Sam Winnall within three minutes.
The U's hit back on fifteen minutes though, with Sanchez Watt scoring for the second time in six days.
A penalty and a header from a corner then put the hosts 3-1 ahead, before Freddie Sears reduced the arrears.
An equaliser couldn't be found and the game ended at 3-2 to Barnsley.
He decided that skipper Sean Clohessy was the best option and that also meant a starting place for returning loanee Elliott Hewitt at right back.
U's boss Tony Humes had to make changes to his starting line up, with injuries to both Magnus Okuonghae and Frankie Kent giving him a selection headache at centre half.
Another change in the back four saw left back Michael O'Donoghue come in for his senior debut, relegating Ben Gordon to the bench.
The home side started brightly and it only took them three minutes to open the scoring.
Winnall was able to pull out onto the angle to receive a forward pass, and drilled an excellent finish into the bottom corner.
Another attack down the right for the Tykes led to a Cole cross shot but Elliott Hewitt was in the right place not once but on three occasions to clear.
It was looking like the U’s were struggling to contain Barnsley but in the fifteenth minute, they quickly turned defence into attack and got an equaliser.
A Barnsley move was halted and David Fox clipped a ball out to Elliott Hewitt on the right.
The Ipswich Town loanee pulled a clever cross back along the eighteen yard line and Sanchez Watt hit a shot that looked like it took a slight deflection on its way into the corner.
The home side thought they had got themselves in front again in the twenty sixth minute but the offside flag was up before the header went into the top of Sam Walker’s net.
A minute later, and the U’s almost got in front, as George Moncur found Sears on the right and his angled drive had to be pushed to safety by Turnbull.
Both sides then had long range efforts, with Hourihane and Hewitt failing to test the opposition keeper.
Goalscorer Winnall then shot over the U’s bar, before a strong penalty claim by the home team was waved away.
Sears gave away possession with a stray cross field ball and in his efforts to make amends, put in a challenge in the box that had the home supporters, and players for that matter, appealing to the official.
The final chance of the half fell to Sears but the U’s topscorer couldn’t find the target.
With the scores level at 1-1, both sides came out strongly in the second half and the opening minutes were played at a frenetic pace.
Neither keeper was really tested though but both sides looked like they were looking for goals.
And it was Barnsley who took the lead again, scoring from the spot after a foul by captain Sean Clohessy.
Cole took a pass superbly in his stride and the referee decided that Clohessy’s sliding challenge took the man rather than the ball and pointed to the spot.
Hourihane stepped up and calmly sent Walker the wrong way to put his side 2-1 up with fifty four minutes played.
The U’s were trying hard for another equaliser but Moncur, Gilbey and Sears all had efforts comfortably saved by Turnbull in the home goal.
Those saves proved important for Barnsley, as they increased their lead on seventy minutes.
An outswinging corner found its way to the far post and Ramage climbed above Gilbey to plant his header back beyond Walker to make it 3-1.
Immediately after the goal, Humes bought on Rhy Healey for Massey, and the Cardiff City loanee made an instant impact.
He ruffled a few feathers in the Barnsley defence with a couple of boisterous challenges, before setting up Sears for a second U’s goal.
Fox and Healey exchanged passes before the latter threaded a ball into Sears’ path – and the U’s top scorer took a touch before burying his right foot effort past Turnbull.
Kemar Roofe and Sammie Szmodics were both introduced as the U’s looked to draw level in the final minutes but under pressure, Barnsley held on.
Roofe provided the best threat late on though, hitting a couple of teasing crosses and heading into Turnbull when well placed at the far post.
The four minutes of injury time weren’t enough for the U’s to get an equaliser and the final whistle went on a 3-2 defeat for Humes and his squad.