The unified world champion, who holds the IBF, WBA and WBO titles, will tackle the Ukrainian live on Sky Sports Box Office in the UK and Ireland.
Joshua (24-1, 22 KOs) is coming off a devastating knockout victory over Kubrat Pulev, dispatching the Bulgarian in the ninth round at The SSE Arena in Wembley last December.
The 31-year-old was primed to fight fellow Briton and WBC world heavyweight champion Tyson Fury to crown an undisputed heavyweight king.
The Gypsy King has been forced into a rematch with Deontay Wilder, with the bout since postponed due to contracting Covid-19.
Usyk is Joshua’s mandatory challenger and continues to push for a world title at heavyweight after emerging from cruiserweight as the undisputed king.
The home of Spurs will also allow AJ to follow in the footsteps of Frank Bruno, who defeated Joe Bugner at the old White Hart Lane back in October 1987.
Usyk (18-0, 13 KOs), who knocked out Tony Bellew during his spell in the 200lb division, is among the best pound-for-pound fighters in the world and has won both of his heavyweight fights since stepping up.
The Ukrainian, who won heavyweight gold at the same 2012 games at which Joshua became an Olympic champion at super-heavyweight, retired Chazz Witherspoon in seven rounds at the Wintrust Arena in Chicago in October 2019 before outpointing Derek Chisora at The SSE Arena last October.
“The date is set and we are fully locked in,” said Joshua. “September 25, Tottenham Hotspur Stadium, I put it all on the line again and it’s time to defend my crown. We are two Olympic Gold medallists who have fought our way to the top and never avoided challenges.
"The stadium is exceptional, the atmosphere will be electric, I’m honoured to be the first person to fight in such an awe inspiring venue. The stage is set and I am ready to handle business.”
Usyk said: “The path will be mastered by the walking one.”
“Olympic gold vs Olympic gold, unified world heavyweight champion vs undisputed cruiserweight world champion, this one has it all,” said Eddie Hearn, Joshua’s promoter.
“I can’t wait to stage this huge event in front of over 60,000 at the stunning Tottenham Hotspur Stadium on 25 September.”
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