Juventus' shock loss to Young Boys left the door open for United to go through to the last 16 as winners of Group H, but a Carlos Soler strike and a Phil Jones own goal saw them deservedly lose at Mestalla, although Marcus Rashford netted a consolation.
Jose Mourinho made a number of changes to his starting line-up, with their place in the next round secure and a trip to Liverpool on Sunday looming, and the players on show, including Paul Pogba and Romelu Lukaku, did nothing to suggest they will keep their spots for that game at Anfield.
The result means little to Valencia, who were already assured of a Europa League place, but the ease with which they claimed only a second competitive win over United will give them heart in a difficult season.
Valencia went ahead with the first shot on target of the match, Soler drilling clinically into the bottom-left corner of Sergio Romero's goal after Jones had headed a cross straight to him.
Juan Mata had United's first effort of the game 26 minutes in and was unlucky not to win a penalty, with Mouctar Diakhaby clearly blocking the goal-bound shot with his arm, before Paul Pogba somehow prodded wide with the goal at his mercy following a corner.
With no shots on target before the break, United would have hoped for a strong start to the second half, but they were 2-0 down 47 minutes in when Jones diverted a throughball past the onrushing Romero.
Substitute Rashford finally tested Valencia goalkeeper Jaume Domenech 72 minutes in, and Andreas Pereira was unlucky to see a stinging effort clawed away by his former team-mate, as United at last put the home side under pressure.
Rashford met Ashley Young's cross with a firm header with three minutes of normal time left, but Juan Mata failed to connect with a clever Pogba chip as the chance to rescue a draw came and went.
What does it mean? United limp through but glaring concerns persist
United did not expect a favour from Young Boys against Juventus to help them win the group, but their performance at Mestalla was so abject that the group's other shock result was entirely redundant.
Mourinho had always planned to rest several first-choice players, but if he hoped for others to give him a selection headache ahead of a packed league schedule, he was sorely disappointed.
United are in the next round, but not many group winners will fear them in next Monday's draw. Valencia, meanwhile, can take some heart into their Europa League campaign.
Shining Soler proves his point
Soler spoke of his annoyance that United secured their last-16 spot with a controversial Marouane Fellaini winner against Young Boys, and he played here like a man determined to show his team deserved to go through. He was so dangerous in the first half that Marcos Rojo was hauled off at half-time.
A day for Jones to forget... and quickly
Nobody impressed in a red shirt, but Jones must bear the brunt of the criticism. Having set up Soler for the opening goal with a tame defensive header, he scored Valencia's second himself with an errant sliding challenge.
Key Opta Facts
- Valencia have beaten United in a Champions League match for the very first time in what was the eighth meeting between the sides in the competition (W1 D5 L2).
- United (three wins) have progressed to the knockout stages of the Champions League with no more than three group-stage victories for the first time since 2008/09 (2), a campaign in which they went on to reach the final.
- Jones is the first United player to score an own goal away from home in a Champions League match since Mark Lynch did so against Deportivo La Coruna in March 2003 – his only senior appearance for the Red Devils.
Valencia travel to Eibar in their next LaLiga match on Saturday. A day later, United face that daunting trip to Anfield.