A little over a fortnight ago, the Catalan giant had a nine-point lead and was also favorite to defend its Champions League crown, but a sudden loss of form has changed all that.
Valencia's 2-1 win is a complete reversal of the result the last two times the side's met in the Nou Camp, Barcelona winning 7-0 in the Spanish Cup.
It was one of a series of setbacks which led to the sacking of English coach Gary Neville, but despite a recent improvement, few were expecting Valencia to offer much resistance in the latest meeting.
But after soaking up some intense early pressure, it was Ivan Rakitic's deflected own goal and a Santi Mina strike just before halftime that set the visitor on its way to an unlikely three points.
Lionel Messi snapped his recent goal drought, his first in five games, in the 63rd minute in a flowing move.
It was his 500th goal for club and country and gave Barca renewed hope.
Excellent chances fell to Rakitic and finally Gerard Pique, who screwed his late effort wide and put his head in his hands.
Barcelona is still top of La Liga, but only only goal difference from Atletico Madrid, which made no mistake earlier Sunday with a 3-0 home win over lowly Granada, with Koke, Fernando Torres and Angel Correa on target in the Vicente Calderon.
Arch-rival Real Madrid is also only a point behind in third place after its 5-1 trouncing of Getafe Saturday,
with all three teams having five games to play.
It was Real's 2-1 victory in El Clasico on April 2
, despite being reduced to 10 men, which has proved the catalyst for Barca's sorry run.
A 2-1 first leg victory over Atletico in their Champions League quarterfinal tie was a good response, but a 1-0 defeat at Real Sociedad followed.
A devastating 2- 0 defeat in the second leg to Atletico saw Barcelona's tenure as European champion ended and now the latest setback.
It is the first time since 2003 Barcelona has suffered three La Liga defeats in a row, with a midweek game against Deportivo Coruna now taking on added significance with pressure mounting.
Defiant coach Luis Enrique said his team would pick itself up after the latest defeat. "We were the better team, we put in a complete performance," he said.
"We have to pick ourselves up. We will face the challenge as a Barca player does, with his head held high."