Darlington claimed their first victory in five matches to end Cambridge's 10-game unbeaten league run.
The U's, who dropped out of the play-off places, went behind when Adam Rundle shot past Danny Naisbitt from the edge of the penalty area.
Cambridge nearly equalised in the second half but Harrison Dunk's powerful effort bounced off the post.
Darlington capitalised on that miss as Adam Reach headed in from Rundle's fine cross to secure the three points.
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Cambridge United boss Jez George tells BBC Radio Cambridgeshire:
"I thought there were spells of the game where we did get to grips with it and we did hit the post.
"But it was a demonstration from Darlington of how to press the ball for the entire 90 minutes.
"It never quite happened for our wingers, despite a couple of decent situations for us."