Barcelona were denied the chance to open up a three-point lead at the top of La Liga after Valencia won 2-0 on Saturday.
Jordi Alba scored an own goal, while Maxi Gomez also found the net for Los Che at the Mestalla to slow down Barca's momentum in the title race. It's a first defeat for Quique Setien since he replaced Ernesto Valverde in the Barcelona dugout.
The Blaugrana will lose top spot if nearest rivals Real Madrid avoid defeat in Valladolid on Sunday.
A rather drab first half saw Valencia have the better of the play. Los Che's reward was a penalty when Gerard Pique felled Jose Luis Gaya in the area.
Gomez stepped up but found himself denied by Marc-Andre ter Stegen. It was far from the last time Barca's outstanding goalkeeper would be called into action.
The hosts also went close when former Arsenal holding midfielder Francis Coquelin tried his luck from distance. It was an ambitious effort, but Los Che created better chances whenever Carlos Soler got on the ball.
Soler teed up both Kevin Gameiro and Gomez to continue the hosts' control of the game. Yet Ter Stegen still refused to be beaten.
Barca's only response was to try to get Lionel Messi into shooting space. However, the Blaugrana's attacking talisman wasn't seeing enough of the ball.
The visitors' sluggish display was suitably punished three minutes after the restart when the unfortunate Alba helped turn another effort from Gomez into his own net:
Barca lacked energy and aggression, so Setien introduced Arturo Vidal for Arthur Melo at the heart of midfield. A little more impetus led to more chances for Messi, who was played in twice by Alba and sent clear by Antoine Griezmann, only to be frustrated by last-ditch defending and wayward finishing.
An equaliser seemed inevitable, but it was actually Los Che who doubled their lead against the run of play. The goal came in the 77th minute when the lively Gomez finished smartly after meeting a pass from Ferran Torres.
Ivan Rakitic was sent on late for an overwhelmed Frenkie de Jong, with Barca hoping to make better use of possession. However, despite him playing Messi in for yet another chance, the Croatian playmaker's introduction did nothing to alter the outcome.
Defeat offers a reminder of the work Setien needs to do to keep Barca on track. Luis Suarez being on the shelf has left the attack overly reliant on Messi, while there's still not enough creativity nor the right balance in midfield.
Both teams have Copa del Rey duty in the week, with Valencia away to Cultural Leonesa in the last 16 on Wednesday. Meanwhile, Barca host Leganes a day later.