Justin Timberlake told the audience that he meant "no disrespect" before a recent rendition of his song Cry Me a River.
The SexyBack singer seemingly alluded to the backlash he has received following the release of his ex-girlfriend Britney Spears's memoir, The Woman in Me, in October.
According to video footage, Justin told the crowd, "No disrespect," before launching into the 2002 breakup song during his hour-long set at the opening of the Fontainebleau in Las Vegas on Wednesday night.
The disclaimer comes months after he told the audience at Dave Chappelle's birthday party in August, two months before the book's release, that he would never stop performing the song.
"They said not to do this song no more. F**k that!" he said at the time.
The singer dated Britney from 1999 to 2002, when the couple broke up amid cheating rumours.
Cry Me a River features lyrics about infidelity and the accompanying music video starred a woman who looked like Britney.
The former NSYNC star has not publicly addressed the bombshells in the pop singer's autobiography. In the book, she claimed he had been unfaithful during their relationship and that she fell pregnant while they were dating and she got an abortion because he wasn't ready to be a father.
She also confirmed a long-running rumour that she cheated on Justin by kissing choreographer/dancer Wade Robson.
Justin is now married to actress Jessica Biel, who was in the audience at his Las Vegas show this week.