Usyk almost stopped the Briton in the final round, but took a unanimous points decision to join Evander Holyfield and David Haye as cruiserweight champs who have gone on to world title glory at heavyweight.
The Ukrainian won by margins of 117-112, 116-112 and 115-113 to take Joshua's WBA, IBF, WBO and IBO belts after a quite superb display against his fellow Olympic boxing champion from London 2012.
Usyk shaded the early exchanges in just his third heavyweight contest, using his speed to score regularly with one-twos from his southpaw stance.
In a fascinating technical encounter, the first real fireworks came at the end of round three when Joshua was troubled by another Usyk one-two before landing a powerful right just before the bell.
The home favourite struggled to handle the challenger's accuracy and range of shots in the first half of the fight, but a couple of right hooks late in round six gave the crowd something to shout about.
That was the spark for Joshua to commit more to attack, but Usyk proved hard to hit and stopped the champion momentarily in his tracks with a one-two late in round seven.
It soon became clear that the champion needed something special to change the course of the contest, but Usyk took his punches well and landed enough shots of his own to stay ahead on the scorecards.
Much had been made of Usyk's lack of a knockout punch, but he had enough power to dissuade Joshua from launching reckless attacks in the closing stages of the fight.
Indeed, it was the visitor who almost finished it inside the distance with Joshua - who suffered a suspected eye-socket fracture - doing well to survive a flurry of shots late in round 12.
At the final bell, Joshua went straight to his corner for treatment as Usyk knelt and crossed himself in the middle of the ring.
Usyk was undisputed world champion at cruiserweight before moving up a weight division two years ago.
He now has a 19-0 record, and a unification fight with the winner of next month's WBC title bout between Deontay Wilder and Tyson Fury awaits.
Speaking via an interpreter, Usyk said in his post-fight interview, "This means a great deal to me. The fight went exactly the way I expected it to go. There were a couple of moments where Anthony pushed me hard but nothing extraordinary."
When asked about whether Joshua would invoke a rematch clause, he said, "I've been working so hard since January. I haven't seen my family for so long. I want to go home and spend time with my family. I'm not thinking about a rematch."
This was an 11th consecutive world heavyweight title fight for Joshua who slips to 24-2, and his future is uncertain after Usyk showed exactly he is considered one of the best pound-for-pound boxers in the world.
In the main support bout, Lawrence Okolie made a successful first defence of his WBO cruiserweight belt against the previously unbeaten Dilan Prasovic.
An Okolie right hook sent the Montenegrin challenger to the deck but he managed to get up and complete the round.
It was all over in the next with Prasovic unable to beat the count after a crunching left to the body from the Londoner.
Rio 2016 Olympian Okolie has now won all 17 of his professional contests and said afterwards he was hoping for a unification bout, preferably with Latvia's IBF title holder Mairis Breidis, before becoming undisputed champion and then moving up to heavyweight like Usyk.
Callum Smith made a spectacular debut at light-heavyweight with a second round knockout of Dominican Republic's Lenin Castillo.
A vicious right hook sent Castillo to the canvas with his legs twitching horribly in response to the shot. Promoter Eddie Hearn said on the broadcast that "he's in hospital for precautions, but he's fine which is fantastic".
Liverpool's former WBA super-middleweight champion Smith, whose sole defeat came at the hands of Canelo Alvarez last December, looks sure to be fighting for world titles soon in this higher weight class.