Lionel Messi’s 500th career goal could not prevent stumbling leaders Barcelona suffering a third successive La Liga defeat for the first time since 2003.
Ivan Rakitic deflected Guilherme Siqueira’s cross-shot into his own net to give Valencia a shock lead.
Santi Mina then finished a terrific move by slotting in a second goal in first-half stoppage time.
Messi ended a club goal drought of 515 minutes, but Barcelona now only lead Atletico Madrid on goal difference.
This was some transformation for a Valencia side humiliated 7-0 on their last visit to the Nou Camp, in the first leg of a Copa del Rey semi-final on 3 February.
Pako Ayestaran, who replaced the sacked Gary Neville as manager on 30 March, put out a line-up with far more discipline and spirit than they showed on that miserable night.
Shkodran Mustafi, once part of the youth set-up at Everton, was pivotal to their victory. The German centre-back was outstanding in frustrating Barcelona’s much-vaunted forward line for much of the evening – as Ayestaran’s side climbed to 12th place.
Siqueira, on loan from Barcelona’s title rivals Atletico Madrid, also had a significant role to play for Valencia.
The Brazilian full-back broke forward on to Andre Gomes’ pass and looked as if he would be credited with the opening goal as his delivery from the left was fumbled in at the near post by Claudio Bravo. Television replays, though, showed that Rakitic had got the decisive touch.
Mina, who scored when the reverse fixture ended 1-1 in December, then took advantage to place Dani Parejo’s through pass inside Bravo’s far post as Sergi Roberto failed to track his run.
Luis Enrique’s side began April with a nine-point lead over second-placed Atletico, but their season has crumbled since then, with defeat against Real Madrid in El Clasico marking the start of a dreadful run.
It was Atletico who ended Barcelona’s chances of retaining the Champions League in midweek, and who drew level at the top of La Liga with a comfortable 3-0 victory over Granada earlier on Sunday.
Barca, playing the 53rd match of a draining season that included a trip to Japan in December to win the Club World Cup, looked weary and short of ideas as they attempted to fight back from a 2-0 half-time deficit.
They could lose top spot if they fail to win at Deportivo La Coruna on Wednesday, with Atletico visiting Athletic Bilbao on the same night, and Real Madrid – just a point behind in third place – also in action, at home to Villarreal.
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