Holzhauer earned $131,127, breaking the record of $110,914 he set back on April 9.
He now owns the top four spots for single-game winnings on "Jeopardy" and is second in total winnings, trailing only Ken Jennings,
Jennings won 74 games in a row, winning more than $2.5 million in 2004. So far, Holzhauer has won $697,787.
Holzhauer, 34, is a native of Naperville, Ill. and now lives in Las Vegas, where he works as a professional sports bettor.
Also Wednesday, "Jeopardy" host Alex Trebek posted a video saying he plans to return for a 36th season as he fights cancer.
Trebek is fighting stage 4 pancreatic cancer. He wanted to thank everyone for the messages of encouragement and support, particularly the cards he received from young viewers.