In 1910, former undefeated heavyweight champion James J. Jeffries came out of retirement to challenge Johnson, saying "I am going into this fight for the sole purpose of proving that a white man is better than a Negro". He had not fought in six years and he also had to lose well over 100 pounds in order to get back to his championship fighting weight. Efforts to persuade Jeffries to "retrieve the honor of the white race" began immediately after the Burns-Johnson fight. Initially Jeffries had no interest in the fight, being quite happy as an alfalfa farmer.[citation needed] On October 29, 1909, Johnson and Jeffries signed an agreement to "box for the heavyweight championship of the world" and called promoters to bid for the right to orchestrate the event.

In early December 1909, Johnson and Jeffries selected a bid from the nation's top boxing promoters—Tex Rickard and John Gleason. The bid guaranteed a purse of $101,000 to be divided 75% to the winner and 25% to the loser, as well as two-thirds of the revenues collected from the sales of the right to film the fight (each boxer received one third of the equity rights).

Jeffries mostly remained hidden from media attention until the day of the fight, while Johnson soaked up the spotlight. John L. Sullivan, who made boxing championships a popular and esteemed spectacle, stated that Johnson was in such good physical shape compared to Jeffries that he would only lose if he had a lack of skill on the day of the fight. Before the fight, Jeffries remarked, "It is my intention to go right after my opponent and knock him out as soon as possible." While his wife added, "I'm not interested in prizefighting but I am interested in my husband's welfare, I do hope this will be his last fight." Johnson's words were "May the best man win."

Racial tension was brewing in the lead up to the fight and in order to prevent any harm from coming to either boxer, guns were prohibited within the arena along with the sale of alcohol and anyone who was under the effects of alcohol. Apples were also banned as well as any weapon whatsoever. Behind the racial attitudes which were being instigated by the media was a major investment in gambling for the fight, with 10–7 odds in favor of Jeffries.

The fight took place on July 4, 1910, in front of 20,000 people, at a ring which was built just for the occasion in downtown Reno, Nevada. Jeffries proved unable to impose his will on the younger champion and Johnson dominated the fight. By the 15th round, after Jeffries had been knocked down twice for the first time in his career, Jeffries' corner threw in the towel to end the fight and prevent Jeffries from having a knockout on his record.[26]

Johnson later remarked he knew the fight was over in the 4th round when he landed an uppercut and saw the look on Jeffries face, stating, "I knew what that look meant. The old ship was sinking." Afterwards, Jeffries was humbled by the loss and what he'd seen of Johnson in their match. "I could never have whipped Johnson at my best", Jeffries said. "I couldn't have hit him. No, I couldn't have reached him in 1,000 years."

The "Fight of the Century" earned Johnson $65,000 (over $1.8 million in 2020 dollars) and silenced the critics, who had belittled Johnson's previous victory over Tommy Burns as "empty", claiming that Burns was a false champion since Jeffries had retired undefeated. John L. Sullivan commented after the fight that Johnson won deservedly, fairly, and convincingly:

The fight of the century is over and a black man is the undisputed champion of the world. It was a poor fight as fights go, this less than 15-round affair between James J. Jeffries and Jack Johnson. Scarcely has there ever been a championship contest that was so one-sided. All of Jeffries much-vaunted condition amounted to nothing. He wasn't in it from the first bell tap to the last ... The negro had few friends, but there was little demonstration against him. (Spectators) could not help but admire Johnson because he is the type of prizefighter that is admired by sportsmen. He played fairly at all times and fought fairly. ... What a crafty, powerful, cunning left hand (Johnson) has. He is one of the craftiest, cunningest boxers that ever stepped into the ring. ... They both fought closely all during the 15 rounds. It was just the sort of fight that Jeffries wanted. There was no running or ducking like Corbett did with me in New Orleans (1892). Jeffries did not miss so many blows, because he hardly started any. Johnson was on top of him all the time.... (Johnson) didn't get gay at all with Jeffries in the beginning, and it was always the white man who clinched, but Johnson was very careful, and he backed away and took no chances, and was good-natured with it all ... The best man won, and I was one of the first to congratulate him, and also one of the first to extend my heartfelt sympathy to the beaten man.

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