Dejan Lovren's header eight minutes into the second half was the difference as leaders Liverpool were beaten for the first time this season.
The Croatia international defender met Adam Lallana's left-wing corner and, despite close attention from his marker, managed to divert the ball home with the angle against him.
The hosts made three changes to the side that drew 2-2 with Swansea City on Monday, Iago Aspas replacing the injured Philippe Coutinho and Daniel Agger and Kolo Toure coming in for Andre Wisdom and Jose Enrique in the defence.
Southampton manager Mauricio Pochettino named an unchanged side from the team that drew 0-0 with West Ham last time out.
Jordan Henderson had the first effort on target in the third minute, running on to an Aspas ball on the edge of the box, but his curled effort was straight at Artur Boruc.
After 21 minutes, Boruc pulled off a save of much higher quality, flying to his right to tip away a Steven Gerrard free-kick that was destined for the top corner following Jose Fonte's foul on Aspas.
As the pace of the game intensified, Rickie Lambert wasted a golden chance to open the scoring in the 27th minute, swivelling on a loose ball eight yards from goal, but he failed to make a true connection and his shot looped up into Simon Mignolet's arms.
The visitors were lucky not to concede a penalty five minutes later when Daniel Sturridge looked to have been tripped as he cut inside Lovren, although the referee waved play on.
Boruc once again came to his side's rescue nine minutes before the break when he denied Victor Moses at the near post after the Nigeria international had weaved his way past two defenders on the left-hand side.
On the stroke of half time, Sturridge met Gerrard's cross from the right, but the ball cannoned off the striker's shoulder rather than his head and looped onto the roof of Boruc's net.
Mignolet put his side under pressure two minutes after the break when he tried to play the ball out from the edge of the box, but as Southampton won possession the Belgium international raced off his line to deny Lallana and redeem himself.
In the 53rd minute, it was the visitors who took the lead when Lovren met Lallana's corner and despite being off balance, he managed to power his header between the diving Mignolet and Gerrard on the line.
A minute later it was almost two as Southampton broke and Pablo Daniel Osvaldo's low shot was kept out by a strong hand from Mignolet.
The hosts were stunned into action, Boruc denying Gerrard from another free-kick, this time low to his left in the 59th minute.
But it was Southampton who almost extended their lead, with substitute Steven Davis riding three challenges on a charge into the box before being denied by the legs of Mignolet, the goalkeeper reacting quickly to tip away the rebound as Davis looked to pounce.
Sturridge wasted a good opportunity with 12 minutes left when, after finding himself in plenty of space on the right, the England forward curled the ball high over the crossbar.
Liverpool continued to press but substitute Luis Alberto's weak effort in the 87th minute was blocked as Southampton held on to seal the points.
