Cambridge suffered their first defeat of the season as Matty Brown's second-minute header sealed victory.
The defender, on loan from Chesterfield, scored his first goal for the Port, nodding in from a corner.
The U's had a great chance to draw level on the stroke of half-time, but Ian Miller's unmarked header from six yards went over the bar.
Luke Berry came close to scoring a late equaliser, but his shot from the edge of the box shaved the post.
Southport secured their first win in six matches, and with it ended Cambridge's 16-match unbeaten run.
VIEWS FROM THE DRESSING ROOM
Cambridge United boss Richard Money told BBC Radio Cambridgeshire:
"I think we will play a lot worse than that, and have played a lot worse than that, and win. I thought we had a good go at it and just couldn't find the right pass
"We've had I don't know how many set-plays and on a number of occasions got free headers and still couldn't score.
"In the key moments we weren't quite good enough. We had a lot of chances and on another day you win that game, but we haven't.
Southport 01 Hurst 20 Fitzpatrick 24 Brown 02 Smith 12 Challoner 06 Brown 18 George 08 Milligan 14 Rutherford 23 Ellington 15 Hattersley (Ledsham - 88' ) Substitutes 21 Osawe 10 Ledsham 04 Akrigg 19 Stevenson 05 Flynn Cambridge United 01 Maxwell 04 Coulson 03 Taylor (Dunk - 60' ) 14 Tait 22 Spillane (Berry - 55' ) 06 Miller 08 Champion 16 Austin 17 Hughes (Arnold - 46' ) 09 Cunnington 21 Appiah Substitutes 19 Sam-Yorke 13 Norris 11 Dunk 24 Arnold 18 Berry
Ref: Adrian Holmes