A stunning stoppage time winner from Stevan Jovetic ended Real Madrid's Spanish record 40-game unbeaten run on Sunday as Sevilla scored twice in the final five minutes to win 2-1 at the Sanchez Pizjuan.
Cristiano Ronaldo's penalty put Madrid in front 23 minutes from time, but Sergio Ramos's own goal levelled before Jovetic, making his La Liga debut on loan from Inter Milan, curled home from 25 yards.
Victory takes Sevilla ahead of Barcelona into second and to within a point of Real at the top of the table, but Madrid also have a game in hand.
"I couldn't imagine a better start," Jovetic told BeIN Sports Spain after only arriving in Seville on Tuesday.
Defeat ends a marvellous run for Zinedine Zidane's men, who haven't tasted defeat since losing to Wolfsburg on April 6.
However, the Frenchman urged his players to shrug off their disappointment with Real still well-placed to win a first ever treble of La Liga, Copa del Rey and Champions League.
"We did the most difficult thing which was to score the goal and with such little time left they caught us out twice, which is the disappointing thing," said Zidane.
"I have to be very proud of them all for what we have done until now.
"We know this was going to happen some day, it is today, and now we have to continue working hard in all competitions."
Sevilla had been just seconds away from ending Real's run just four days ago until Karim Benzema's stoppage time equaliser allowed Real to advance 6-3 on aggregate in the Copa del Rey after a 3-3 draw on Thursday.
However, the bad blood from that match boiled over as former Sevilla favourite Ramos's clash with the most fervent section of the home support continued with the Real captain roundly jeered every time he touched the ball.
And the home fans took extra delight as it was Ramos, so often Real's saviour with important late goals, who inspired Sevilla's fightback when he turned into his own net before Jovetic struck in the most dramatic of circumstances.
Until the final five minutes Real had looked defensively solid as Zidane switched to a back three to match up with Sevilla's favoured formation.
And in contrast to Thursday's goal fest, the sides cancelled each other out in an intense first 45 minutes short on clear-cut chances.
Ronaldo had the best opportunity of the first-half when he sliced wide from Benzema's dangerous cross.
However, the match and title race, looked to be swinging decisively in Real's favour when Sergio Rico clipped the heels of Dani Carvajal as the Madrid full-back burst into the area midway through the second-half.
Despite Vitolo's best attempts to damage the penalty spot as Ronaldo prepared to take, the Portuguese comfortably dispatched the ball into the bottom corner for his 23rd goal against Sevilla.
Real's run has been littered with late goals and fightbacks, but for once the European champions were on the receiving end of a stunning turnaround late on.
Firstly, Pablo Sarabia's fiercely dipping free-kick caught Ramos off guard as he powered a header into his own net.
The Spanish international nearly scored another own goal seconds later as he turned Jovetic's dangerous drive over the bar
But Jovetic grabbed his second goal in as many games for the Europa League holders two minutes into stoppage time when he curled the ball into the top corner from despite Navas getting a hand to his effort.