All good things must come to an end.
Word of the shocking defeat started spreading on Sunday when a leaked clip from the episode circulated on social media.
And in a surprising twist, Holzhauer, who often bludgeoned his opponents by doubling and even tripling their totals, lost by more than $22,000 — even though he responded correctly in Final Jeopardy, according to a clip obtained by NBC News.
Challenger Emma Boettcher, a librarian at the University of Chicago, dethroned the Las Vegas pro sports gambler, who many expected to smash former champ Ken Jennings' record of $2.5 million, set over 74 games in 2004.
“I lost to a really top-level competitor,” Holzhauer told The New York Times. “She played a perfect game. And that was what it took to beat me.”
Boettcher led going into the final round with $26,600. Holzhauer had $23,400, according to the video clip.
The final clue read: "The line 'A great reckoning in a little room' in 'As You Like It' is usually taken to refer to this author’s premature death."
All three contestants got the correct answer: 16th century English playwright Christopher Marlowe.
But this time, it was Boettcher who went big, wagering $20,201, bringing her final total to $46,801.
Holzhauer bet an uncharacteristically small amount, just $1,399.
He finished with $24,799 for the game, and was awarded $2,000 for coming in second place, bringing his total purse to $2,464,216.
The crowd was audibly stunned by the turn of events, and even level-headed host Alex Trebek seemed taken aback.
“Oh gosh! What a payday,” Trebek said. “What a game! Oh my gosh!”
Holzhauer, 34, appeared to take the loss graciously, walking over to Boettcher and giving her a high-five.
Holzhauer became a pop culture phenomenon in recent weeks for his relentless buzzer and bet-big strategy.
Excluding tournaments, Holzhauer and Jennings are the only two people to win more than $2 million on the long-running game show. (Champion Brad Rutter owns the all-time mark of $4.7 million, which includes tournament winnings, over the course of 14 years.)
Holzhauer managed to become the show's second all-time biggest moneymaker after only two weeks.
He once won $131,127 to set the single-game "Jeopardy!" record, topping the previous record set by ... himself.
That mark, $110,914, easily eclipsed the previous one-day record of $77,000 set back in 2010.
Holzhauer first appeared on "Jeopardy!" on April 4 and quickly created a buzz around the program about a month after Alex Trebek revealed he has pancreatic cancer, putting a renewed spotlight on the game show.
Interestingly, Holzhauer, who has a 4-year-old daughter, tweeted Sunday night about losing.
"My kid cried about the possibility of her dad losing, so I told her we could have a party the day after it inevitably happens. Now she cries when I win," he wrote.
The show itself embraced Holzhauer, even going so far as to create a tracker to keep tabs on his accomplishments.