The Abu Dhabi club were penalised by the competition committee for having an ineligible player on their roster in a previous game.
The club have appealed the decision.
Before Thursday's game at the Al Nahyan stadium, Wahda had a healthy five-point advantage at the top of the group but the defeat and points deduction mean Al Ain are the new leaders.
Wahda are now on 13 points, one behind their Garden City rivals.
But Thursday's events mean that Al Nasr, on 11 points, also come into the reckoning as top teams from the three groups and the best second-placed team progress to the last-four stage.
Jires Kembo Ekoko's goal on the stroke of half time was enough to give the visitors their victory.
Othman Al Shamsi, the Wahda defender, completely misjudged Ali Al Wehaibi's cross from the right to leave the Congolese forward unmarked and with plenty of time to take aim and fire it past Adel Al Hosani, the Wahda goalkeeper.
Up to then, much of play was confined to the middle of the pitch. Eisa Ahmed's header off a corner the closest to breaking the deadlock. The Wahda defender's effort from the front of the posts flew over the crossbar. Mohammed Al Shehhi followed it up with a weak shot off a pass from Eric Mouloungui.
Wahda introduced their two new signings – Mouloungui, the Gabon international who was returning to action after more than six months following ankle surgery, and Dino Djulbic, the Australian centre-back they signed earlier in the month.
However, they could not do much against a solid and organised Al Ain, the Pro League leaders.
Wahda had more possession and territorial advantage, particularly in the second half, yet they failed to break a resolute Al Ain defence who were content in preserving their one-goal advantage.
Both side were missing key players with Emirati players on national team duty in Friday's Gulf Cup final in Bahrain and Al Ain's Asamoah Gyan, the Ghana captain, taking part in the African Cup of Nations.
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