In a match brought forward to allow Valencia to prepare for next week's UEFA Cup final against Olympique Marseille, the visitors made some uncharacteristic defensive errors as Villarreal went 2-0 up inside the first half hour.
Rafa Benitez's side pulled a goal back 10 minutes before the break but although they had several good chances to equalise in second half they were unable to extend their unbeaten run.
The win pushes Villarreal up into fifth place in the table, but with all their immediate rivals still to play they are unlikely to retain the position over the weekend.
Valencia, who were crowned champions last week following their 2-0 victory at Sevilla, fielded a shadow side ahead of next Wednesday's UEFA Cup final.
Villarreal, who lost to Valencia in the semi-finals of the competition, took full advantage of their opponents' relaxation.
Brazilian striker Sonny Anderson gave his side the lead in the 14th minute after he controlled a long pass from countryman Juliano Belletti with his toe, lined up his shot and guided the ball round reserve keeper David Rangel.
Belletti, who is in the process of finalising a move to Barcelona, went close to extending his side's lead in the next attack when he sent a 20-metre drive thundering against the bar.
But the home side did not have to wait long before playmaker Juan Roman Riquelme made it 2-0 with a classy solo effort.
Picking up a pass from Anderson just outside the area, the former Barcelona player checked inside two defenders before curling a shot past Rangel into the far corner.
Forward Jose Mari should have made it 3-0 just after the half hour but he failed with just the keeper to beat.
That scare finally woke Valencia from their slumber and they pulled a goal back 10 minutes before the break when Argentine defender Mauricio Pellegrino rose headed in from a corner.
Valencia twice came close to levelling the game in the second half but it was a determined Villarreal who emerged with a deserved victory.
The match was preceded by a minute's silence in memory of Atletico Madrid owner and former president Jesus Gil who died on Friday.
