Timberlake performed the hit track at a New Orleans Super Bowl charity benefits - the song which is reportedly inspired by his break-up with Britney.
Before performing the song, he introduced it by saying, "Sometimes in life, you think you found the one. But then one day you find out that she is just some bitch!"
Fans messaged Timberlake over his Twitter account, accusing him of talking about Britney, who apparently cheated on him in 2002 with choreographer Wade Robinson - although neither party has ever confirmed or denied it.
Timberlake wrote on Twitter, "Ok. I see you. Wouldn't disrespect ANYONE personally. Ever. #Relax #ItsBritneyBitch I do love that saying though *with accent* #Respect."
JT live onstage in Italy in 2007
Timberlake performed 'Suit & Tie' live for the first time during the benefit, and was joined by Jay-Z for the song.
He also performed two new songs from his upcoming album, 'Pusher Lover Girl' and 'That Girl'.
Watch a video of Justin Timberlake performing 'Pusher Lover Girl' below:
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