The four-time World Player of the Year extended his record scoring run to 12 matches in the league with a penalty after 39 minutes with Valencia having taken the lead through Ever Banega.
Although Barca won their last league game at home to Osasuna, they lost a fortnight ago against Real Sociedad and this was a lacklustre display at the Mestalla.
Like Real Madrid, who lost to Granada on Saturday, Barca seemed to be suffering the effects of their 1-1 Spanish Cup semi-final first-leg but they still have a significant lead over Atletico.
A second-half Diego Costa header saw Atletico maintain their 100 per cent record at home this season and they are seven points clear of their more prestigious city rivals Real.
Barcelona's assistant coach Jordi Roura admitted his side were not at their best.
"It is certainly true that we arrived after a tough game and they always take their toll. In the first half it was difficult for us to get into the game and we improved after that," said Roura, standing-in for Tito Vilanova who is having cancer treatment.
"We came with the intention of picking up three points but it is a difficult stadium to win at and generally I think the draw was fair. In five years we have won only once here.
"In the second half we had more depth and we had three or four chances which we didn't take."
Barcelona rested captain Carles Puyol as former Livepool player Javier Mascherano was thrown into central defence alongside Gerard Pique while Andres Iniesta was once again chosen in attack instead of David Villa and Alexis Sanchez.
Apart from a 5-0 thumping by Real Madrid, and their elimination by the same side in the domestic Cup, Valencia have been in good form with four wins from five matches in the league.
While Barca probed in typical fashion looking for an opening, Valencia broke with speed down the wings with Jose Guardado and Juan Bernat.
Guardado had a snapshot which which went wide in an even contest and another break from the home side saw Roberto Soldado brought down by Mascherano after 22 minutes while the resulting free-kick was fired into the wall by Tino Costa.
Valencia were snapping at the heels of the Barca midfielders who were looking uncomfortable and Valencia got the breakthrough from Banega.
Pique cleared a cross to the Argentine who had time in the centre of the area to bring the ball down and pick his spot in the corner.
Barca appeared in trouble but then they won a penalty as Joao Pereira clipped Pedro Rodriguez on the edge of the area and Messi finished with aplomb from the spot.
It seemed to inspire Barca who raised their tempo towards half-time but it was Valencia who went nearest to scoring in the dying minutes with a long range strike from Guardado which was palmed away by keeper Victor Valdes.
Fabregas shot wide after a delightfully threaded pass from Messi, but it was Valencia who continued to have the better openings with Soldado failing to hit the target when well placed which was followed by substitute Sergio Canales who was unable to put the former Champions League winners under further pressure.
Villa came on as a substitute but inexplicably missed when in front of goal for Barca and then in the final minutes, Valdes spread himself well to deny Soldado once again.
Malaga could only draw 1-1 with Zaragoza and have now just one win from five games as they compete for a top four finish, while Sevilla beat Rayo Vallecano 2-1 and Real Sociedad were 3-0 winners over Mallorca.