Cape Town - Stoke midfielder Charlie Adam damaged Southampton's challenge for a European place as his late strike secured a 2-1 win at the Britannia Stadium on Saturday.
Ronald Koeman's side had hoped to climb above Liverpool into fifth place in the Premier League and also put pressure on Manchester City as the race heats up for places in the Champions League and Europa League.
They made the perfect start when French midfielder Morgan Schneiderlin opened the scoring midway through the first-half.
But Mame Diouf levelled soon after the interval and Scotland midfielder Adam struck with six minutes left to hand Southampton their second defeat in their last three matches.
To make matters worse for Southampton, defender Toby Alderweireld needed an oxygen mask as he was carried off on a stretcher after a nasty fall in the final moments.
Sixth placed Saints are now five points adrift of City and one behind Liverpool with six games remaining.
Koeman's team appealed in vain for an early penalty when Sadio Mane went down under a challenge from Stephen Ireland, then Graziano Pelle fired into the side-netting as the visitors pressed for the opener.
After dominating the early stages, Southampton finally got the goal their pressure deserved in the 22nd minute when Schneiderlin met Jose Fonte's header and tapped in from virtually on the line.
Steven N'Zonzi had a golden opportunity to equalise on the stroke of half-time when the ball dropped to him after a goalmouth scramble, but the Stoke midfielder could only scuff wide from close-range.
Mark Hughes's side drew level two minutes into the second half thanks to a moment of indecision from Southampton goalkeeper Kelvin Davis.
When N'Zonzi chipped a pass into the penalty area, Davis misread the bounce and the ball ricocheted onto the crossbar before falling perfectly for Senegal striker Diouf to lash into the empty net.
Mane led the Southampton response and he almost created a goal for Dusan Tadic after skipping away from two defenders and picking out the Serb, whose weak shot was cleared off the line.
Yet it was Stoke who had the last word as Adam, on as a half-time substitute, bagged the winner in the 84th minute.
Saints claimed Ryan Shawcross was offside, but, after Jon Walters had a shot blocked, Adam's deft touch bought him just enough time and space to send a bobbling shot trickling into the net.
Peter Crouch had a chance to wrap up the points late on, but the former Liverpool striker produced a candidate for miss of the season as he sliced wide of an open goal.