The result of the match in Riyadh on Tuesday leaves the title-holders' hopes of advancing to the last 16 in jeopardy.
The win was Al-Nassr's third in four matches in the league phase of the continental championship and moves Stefano Pioli's side up to third and within two points of compatriots Al-Hilal and Al-Ahli, who lead the standings at the halfway point.
The top eight in the 12-team groups in both west and east Asia advance to the last 16 in March and Al-Ain, who defeated Al-Nassr last season on the way to the title, are in danger of missing out on a place in the knockout rounds.
Hernan Crespo's side have picked up a solitary point in their four games so far and will need a significant turnaround if they are to climb off the bottom of the table in the second half of the league phase.
The side from the United Arab Emirates were comprehensively outplayed throughout, with Anderson Talisca putting the home team in front in the fifth minute with a low drive from distance that slid past Khalid Eisa.
Ronaldo doubled that advantage 26 minutes later when Eisa spilt Sadio Mane's shot from outside the area and the Portuguese striker pounced on the loose ball.
Six minutes later Brazilian winger Angelo Gabriel cut in from the left to hit a shot towards goal that beat Eisa via a deflection off Fabio Cardoso, giving the keeper little chance.
Al-Ain pulled one back 11 minutes after the restart when Park Yong-woo's drive beat Bento when it struck the post and hit the Al-Nassr goalkeeper on the back to cross the line.
But Wesley scored nine minutes from time to kill off any hope of a comeback, and Talisca hit his second of the night in injury time to complete the rout.
Al-Rayyan climbed off the bottom of the table earlier in the evening when the Qatari side picked up their first points of the campaign with a 1-0 win over Pakhtakor of Uzbekistan.
A long punt forward from near the halfway line in the 51st minute by Hazem Shehata, intended for the head of Brazilian forward Roger Guedes, flew over goalkeeper Vladimir Nazarov and into the top corner to give Al-Rayyan victory.