Second-half goals from Mahmout Trezeguet and Roger Guedes saw Al-Rayyan fight back from going behind to Alejandro Romero's 42nd-minute opener for the home side, who needed to win on Monday night to harbour any remaining chance of reaching the knockout phase.
The loss was Al Ain's fifth in seven games and, after Iraq's Al-Shorta drew 1-1 with Esteghlal of Iran in Tehran, the reigning champions sit bottom of the 12-team table on two points.
Sides finishing in the top eight in the league phase in both west and east Asia progress to March's knockout rounds.
The league phase in the west has been dominated by clubs from Saudi Arabia and that continued as Al-Ahli handed Qatar's Al Sadd a 3-1 defeat to remain in first place, while third-placed Al-Nassr won 4-0 against Al Wasl of the UAE.
A bolstering header by Ronaldo to bring his — #ACLElite | #ACLTwo (@TheAFCCL) #ACLElite tally to 6️⃣ this season!#NSRvWAS pic.twitter.com/5SOf6GZbM7February 3, 2025
Roberto Firmino, Roger Ibanez and Riyad Mahrez were on target for Al-Ahli, who fought back from going behind to an early goal by reigning Asian Player of the Year Akram Afif.
Portuguese star Cristiano Ronaldo, who turns 40 on Wednesday, scored twice after Ali Al-Hassan had given Al-Nassr the lead in Riyadh and Mohammed Al-Fatil added a late fourth for a side who, like Al-Ahli, have already booked their berth in the next round.
Al Ain's elimination comes nine months after the club claimed the continental crown for the second time, defeating Japan's Yokohama F. Marinos over two legs in the final in May.
Al-Shorta's draw with Esteghlal, which came as Rewan Amin cancelled out Joel Kojo's fourth minute opener for the Iranian hosts, keeps the Iraqis' slim chances alive until Tuesday, when Persepolis take on Al Hilal and Al-Gharafa play Pakhtakor.
The eastern zone, involving featuring Australia's Central Coast Mariners, resumes next week.
