Arsenal were stung by a late goal from Southampton's Shea Charles as the Premier League leaders fell to defeat in the FA Cup quarter-finals.
The match was Arsenal's first after their Carabao Cup final loss to Manchester City and followed an international break which was notable for 11 Gunners players withdrawing from their national teams because of injury.
However, Mikel Arteta was still able to name a side containing captain Martin Odegaard, Kai Havertz, Ben White and Gabriel Magalhaes, with Arsenal eager to clear this hurdle.
Despite Arsenal having such senior players in their ranks, and dominating possession, it was Southampton who were the side to create the better chances.
They were duly rewarded, with exuberant celebrations breaking out at full-time at St Mary's.
Now a Wembley trip awaits Southampton in the semi-finals, with Arsenal's hopes of a league and cup double crushed.
Championship promotion hopefuls Saints were on a 14-match unbeaten run coming into this game and their plan to get the ball forward quickly and behind the Arsenal defence caused the Gunners problems throughout.
It was from a floated ball into the penalty area that the home side took the lead in the 35th minute.
England defender White misjudged the flight of James Bree's delivery from the right, and that allowed Ross Stewart to control and swiftly plant a sweet finish past Kepa Arrizabalaga and into the right corner.
Arsenal manager Arteta made a triple change in the second half, with Viktor Gyokeres, Noni Madueke and Riccardo Calafiori coming on, but Southampton continued to threaten.
Tom Fellows flashed a ball just over the top of the Arsenal goal before the dangerous Leo Scienza hit the crossbar with a curling effort.
The visitors drew level when Gabriel's clever pass put Havertz through into the area and he cut the ball back for Gyokeres to level in the 68th minute.
But Southampton were not to be denied, and substitute Charles sent the home fans wild as he took on a short pass from Fellows and finished smartly into the bottom left corner.
Arsenal searched for a second equaliser but found it was beyond them, as Southampton reached the semi-finals for the first time since 2021.
This was always going to be a difficult game for Arsenal on the back of their Carabao Cup final loss, with obvious fitness issues in their ranks and Southampton in fine form.
All the same, this was a poor performance from Arteta's side.
They did not create many serious opportunities and it was often 16-year-old Max Dowman who provided the most threat when the Gunners were going forward.
Arsenal never quite got to grips with Southampton's plan to play long balls behind the defence, and the fact key centre-back Gabriel Magalhaes went off with an injury just compounded a bad night.
Another trophy opportunity slipped away with this result, and Arsenal will be perceived by many to be wobbling.
They must refocus. Arsenal remain in a commanding position going into the closing weeks in the Premier League, nine points clear of Manchester City, and they travel to Portugal in the coming week to play Sporting in the quarter-finals of the Champions League.
Arteta's challenge is to ensure that costly back-to-back defeats do not signal the start of an even more serious slump that will leave his team without a trophy of any kind come the end of the campaign.
Southampton were full of confidence coming into this match owing to their excellent recent form.
The home side did not sit back and look to frustrate Arsenal but played with purpose from the first minute.
They pressed the Gunners backline and were not afraid to kick the ball into space to release the pacy Fellows and fellow winger Scienza.
They were not fortunate to beat the Gunners and were the better side throughout the game, having chances to add further goals as both wingers went close in the second half.
They are wearing a yellow kit in this season's FA Cup to commemorate the 50-year anniversary of the last time they lifted the trophy in 1976 and will hope they can create their own historic moment this time round.
Arsenal are next in action in the Champions League when they play Sporting on Tuesday (20:00 BST).
Southampton travel to play promotion rivals Wrexham on the same night in the Championship (20:00 BST).