They beat the Catalan giants 1-0 at the Vicente Calderon on the night thanks to Koke's early strike in the Champions League quarter-final, second leg.
In a breathless opening to the game, Diego Simeone's Atletico side struck both posts and the crossbar.
They took the lead through the Spanish midfielder's fifth-minute strike after good work by David Villa and Raul Garcia.
Barca never found any consistent attacking threat in the second half - with Lionel Messi largely ineffective - to send the Catalans to their earliest exit in seven years.
The four-time winners of the competition may yet also lose out to Atletico for the Primera Division title - the play each other on the final day - and will do so having suffered a first loss to the capital club in four years.
Atletico's start was so compelling that the only shock was they were just 1-0 up after the first 20 minutes.
Koke grabbed the vital goal to send them ahead in the tie after five minutes, although it could have been so much better, but for hitting their hat-trick of near-misses.
David Villa was the chief culprit, spurning two glorious chances, to build on Koke's early score.
Even that early goal relied on the frame being rattled first, as Barca failed completely to recover after Adrian Lopez thrashed against the right-hand post.
Villa was allowed to dink a cross back across goal and Adrian leapt highest to knock the ball down for an unmarked Koke to side-foot home first time from inside the six-yard box.
Barca were rattled and Villa should have punished his former employers even further when Atletico won the ball on the edge of the area only for the striker to shoot into the left-hand post.
Villa made it the complete set when he hooked a shot over Jose Pinto and on to the crossbar after again easily finding a route behind the Barca defence.
They were the type of chances Costa would have been expected to gobble up.
In any case, Barca's minds were seemingly already frayed to breaking point and goalkeeper Pinto's inexplicable decision to try and dribble out of his six-yard box under pressure from Adrian, before making a lucky escape, hardly settled the visitors.
When Barca eventually did venture forward even Messi appeared tentative, heading a Dani Alves cross wide when he found a gaping hole in the home defence.
Barca did at least start to find a foothold for the remainder of the half, albeit without offering any real threat they would score the goal they now required.
The visitors took just two minutes of the second half, though, to create their best chance of the night.
Xavi rolled a perfectly-weighted pass for Neymar, who took a touch, only for Thibaut Courtois to get out early and push the ball away under the Brazilian's feet.
Courtois needed some luck moments later, though, when he lost balance and Messi blazed over.
Atletico went quiet until Gabi wasted a glorious chance on the counter-attack after 70 minutes.
Barca's defenders backed off him as he ran into the area, preferring to mark Villa's run into the middle, and it proved the correct call when the skipper's weak shot was kept out by Pinto's extended leg.
Moments later Atletico had a strong shout for a penalty, when Javier Maschaerano crashed into the back of Villa, but referee Howard Webb waved play on.
The home side's failure to kill Barca off was increasing the tension inside the Vicente Calderon and when Neymar headed narrowly wide the home fans held their breath.
Still Atletico failed to find the killer second goal and Pinto made another sharp stop to deny Cristian Rodriguez in stoppage-time.
Eventually, however, Webb's final whistle came to the delight of the home fans.