In a fan-recorded video, Puth was singing his duet with Selena Gomez – Bieber's ex girlfriend – when he declared, "F--- you Justin Bieber."
The 24-year-old then continued on into the chorus of the single "We Don't Talk Anymore."
Later, Puth appeared to apologize for the comment – which he called a joke – in some since-delated Tweets.
In a screengrab shared by the video's original poster, Puth appeared to write on the social media site, "Hey everyone. I feel like I need to clear something up."
He continued, "What I said at the concert last night about Justin was meant to be a joke."
"However... the way it was said mid song did not sound like one," he wrote. "I am a massive @JustinBieber fan and I have nothing but great things to say about him and his music always."
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Gomez and Puth sparked relationship buzz when they first worked together on "We Don't Talk Anymore," but the singer later said he was single.
"I'm good friends with her now, and we talked about wanting to collaborate, and I was finishing the song, and I was like, 'Wouldn't it be cool if you sang the second verse?' And that's really how it happened. She came over; she recorded it," Puth previously told PEOPLE of the recording process. "I didn't even have a studio set up: She recorded it in my closet, and it really added a lot to the record."
Bieber for his part has not commented on Puth's shout-out during his show. Reps for Puth did not immediately respond to a request for comment from PEOPLE.