First-half goals from Jay Rodriguez and Rickie Lambert either side of John Terry's header ensured Saints moved up to 13th with a deserved victory despite late Blues pressure.
The result sees Chelsea drop to fourth in the Premier League table and they remain in danger of dropping out of the Champions League places with Arsenal now just two points behind in fifth.
Southampton started the brighter and had two early chances with Rodriguez seeing a sidefooted effort saved comfortably by Petr Cech before Branislav Ivanovic blocked well from Lambert.
But Ivanovic was caught out by quality movement from Rodriguez for the opener when he played a crisp one-two with Steven Davis before slotting the ball calmly past Cech to give Saints the lead.
Fernando Torres, again preferred to Demba Ba in attack, was struggling to get in the game at the other end but did have a goal correctly ruled out for handball shortly afterwards.
The European champions were level just after the half-hour mark when Terry headed home a corner from Marko Marin after Jos Hooiveld had slipped to allow the Blues skipper space.
This was only Terry's second Premier League start under Rafael Benitez but any hopes the goal would kick-start Chelsea's afternoon were soon dashed as they were behind again within minutes.
Saints were awarded a free-kick in a good position and Lambert whipped the ball over Torres in the Chelsea wall, and Cech was unable to scramble to keep it out of the net.
Kelvin Davis replaced the injured Artur Boruc in the Southampton goal at the break and the veteran was forced into a good save from Victor Moses on the hour mark.
Saints remained a threat at the other end and Rodriguez could have doubled the lead after outwitting Ivanovic down the left channel but saw his left-footed effort parried by Cech.
It was Chelsea pushing for an equaliser in the closing stages after Benitez introduced Eden Hazard to provide some impetus but Southampton held on for the points that ease them towards safety.