Timberlake, 37, was playing the first of two headline sets at the venue this week when he paused to give his backing to the team as they prepare to face Croatia.
In a reference to the chorus of the David Baddiel and Frank Skinner song Three Lions, Timberlake called out: “It’s…coming…home!” prompting screams and applause from thousands of his fans.
Timberlake continued: “Somebody told me the first time I came here that I became an honorary Brit so – it’s…coming…home!”
The Cry Me A River singer then humoured the audience with an impression of impassioned England fans cheering on the players.
With an attempt at an English accent, he cried: “Go on then Kane, go on! Go on then Maguire come on!”
Following laughter from the audience, Timberlake said: “Ok sorry, I thought I was out there for a second!
“No seriously, we love you, we love being here. On behalf of the Tennessee kids, you always show us so much love. Thank you for having us out tonight.”
Timberlake will play the second of his dates at the O2 on Wednesday night – at the same time as England’s big match. His set scheduled to start at 21.00pm, one hour after the game kicks off.
After the O2 shared footage of Timberlake’s football chant on social media, fans asked whether the venue would be showing the match ahead of the singer’s performance.
In response, a post from the venue’s official Twitter profile read: “Please keep you eye on our Facebook and twitter, we hope to have an announcement later today…”
Three Lions is currently at the top of the hit parade for the third time after reaching number one in 1996 and again in 1998.