Watch: Messi's 500th career goal not enough as Valencia stun Barcelona to throw open title race
The top three teams of La Liga - Barcelona, Atletico Madrid and Real Madrid are now separated by just one point.
Barcelona's stuttering title bid suffered another setback when they lost 2-1 at home to Valencia as they registered a third straight La Liga defeat for the first time since 2003.
Luis Enrique's shell-shocked side are level on 76 points with Atletico Madrid, who knocked the holders out of the Champions League in midweek, and remain top only due to a superior head-to-head record.
An own goal by Ivan Rakitic put Valencia ahead after 27 minutes and Santi Mina added a second just before halftime to stun the Barca supporters at the Nou Camp.
Lionel Messi scored his 500th career goal to reduce the deficit after 64 minutes but the struggling hosts, who have squandered a nine-point lead, could not find an equaliser.
Valencia goalkeeper Diego Alves produced a spectacular save to deny Rakitic late on and Gerard Pique agonisingly missed the target at the near post in stoppage time.