The Argentine rifled home from the spot to break another record by scoring for the 12th consecutive La Liga game, cancelling out Ever Banega's opener for the resilient hosts.
Ernesto Valverde had set out to refuse the Blaugrana their usual time on the ball, and it was a tactic that looked to have worked superbly as Ever Banega put the hosts ahead. Lionel Messi equalized from the spot soon after though, scoring in a record 12th consecutive La Liga fixture.
Both teams opened the game with a lot of energy, but Barca looked more threatening as it was largely in control of possession. The visitors were unable to fashion any chances to test Vicente Guaita as Los Che defended in numbers and pressed quickly.
Valencia complemented its enthusiastic defense by playing on the break when it managed to wrest the ball away from Barcelona, with Roberto Soldado keen to make an impact. Midway through the first half the striker's bright run came to an end on the edge of the box due to a trip from Javier Mascherano. Tino Costa struck the free kick, but he failed to get it past the wall, and his team's appeals for a handball on Xavi were ignored.
Los Che's resilience at the back continued to frustrate the Blaugrana, and Valverde's men were rewarded in the 33rd minute. Soldado's cross was headed away by Gerard Pique, but only as far as Banega, who brought the ball down and rifled a low shot past Victor Valdes at his near post.
Barca were handed a route back into the game as Joao Pereira clumsily cut down Pedro in the box in the 39th minute. Messi stepped up to take the penalty and fired an unstoppable effort into the top left-hand corner for his 34th league goal of the season.
The teams went into the break level, but with just five minutes gone in the second half Valencia nearly regained the lead. Andres Guardado's cross was met by Soldado, but he could not guide his header inside of Valdes' post.
The game continued to go from end-to-end, and five minutes later Cesc Fabregas had a chance to put the Catalan side ahead after Messi's defense-splitting throughball, though his shot only rippled the side netting.
Valencia nearly struck after Sergio Canales replaced Banega in the 64th minute, as it did not take long for the former Real Madrid man to make his presence known. From Guardado's cutback, a fantastic, arrowed shot towards looked to be heading for the top corner until Valdes' fingertips turned it wide of the post.
The keeper was once again in fine form in the 89th minute, as a brilliant double save, the second of which came from a Soldado follow-up effort, stopped the hosts from stealing all three points at the death and ruining David Villa's return to his former home ground.