The all-British clash, witnessed by a crowd of 96,000, was a thrilling spectacle that saw Dubois dominate from start to finish.
The 27-year-old Dubois dropped Joshua multiple times throughout the fight, notably early in the first round - from which AJ seemingly never truly recovered - and the defending champion ultimately secured a knockout with a powerful counter right hook, forcing the referee to step in.
Prior to the fight, Dubois was elevated from his position as interim champion after Oleksandr Usyk - who alongside Tyson Fury was in attendance at Wembley - was forced to vacate.
Joshua, 34, was left reeling from the defeat, which marked his fourth loss in 32 professional fights.
With his latest bid to regain the heavyweight crown finding a devastating end, questions will now begin as to whether he should hang up his gloves or continue on.
Joshua himself suggested he's keen to keep fighting, saying in his post-fight interview: "Credit to him and his team, we rolled the dice of success, but we came up short.
"You know I'm ready to kick off in the ring, but I'm going to keep my cool, keep very professional, and give respect to my opponent.
"I'm always saying to myself I'm a fighter for life. We keep rolling the dice.
"I had a sharp opponent, a fast opponent and a lot of mistakes from my end, but that's the game."
Post-fight, Dubois expressed his excitement and determination, telling DAZN: "I've only got a few things to say, are you not entertained?!
"It's been a journey, and I'm grateful to be in this position. I'm a gladiator. I'm a warrior until the bitter end.
"I want to get to the top of this sport and reach my full potential.
"Behind the scenes, the work with my dad. I've been on a rollercoaster run, this is my time, this is my redemption story. I'm not going to stop until I reach my full potential."
Dominant Dubois
The fight was a one-sided affair from the outset, with Dubois dominating Joshua from the opening bell.
In the second round, Dubois swarmed Joshua, landing heavy shots and putting him on the ropes. Joshua was fortunate to survive the round, but the damage was done.
The third round saw Dubois continue his onslaught, landing a powerful left hook that sent Joshua crashing to the canvas.
Despite making it to the bell, Joshua was clearly struggling.
The fourth round was more of the same, with Dubois dominating the action.
Joshua's resilience was admirable, but it was clear that he was being outmatched.
The fifth round proved to be the final nail in Joshua's coffin. Under pressure from a desperate AJ, a well-timed right hook from Dubois sent him to the canvas, and the referee was forced to stop the fight.
