No. 2 Florida is unbeaten no more, the last of three undefeated teams to fall on Saturday.
Tennessee put an end to the surprising Gators' best start in school history with an 80-76 victory.
Florida, 17-1 overall and 3-1 in the Southeastern Conference, had been the only undefeated Division I team left when it took the court after No. 1 Duke and No. 9 Pittsburgh had lost earlier in the day.
The Volunteers (12-3, 3-1) were not intimidated even after losing two in a row at Louisiana State and at No. 4 Memphis earlier in the week.
After all, they had won at then-No. 6 Texas last month, giving Tennessee fans hope that Coach Bruce Pearl was bringing new life into a program that had been mediocre the past four seasons under Buzz Peterson. He and the Volunteers proved they had changed on Saturday.
The final 9 1/2 minutes were tight and got tighter as the clock wound down.
Florida's Corey Brewer tied the score at 76-76 with two free throws with 45 seconds left. Tennessee's Chris Lofton missed a three-point shot, and Brewer got the outlet pass and was racing for the basket when Lofton stole the ball.
Dane Bradshaw scored a layup that put the Volunteers ahead, 78-76, with 20 seconds left.
Florida's Lee Humphrey then missed a three-point shot with six seconds left, and Lofton was fouled. He made both free throws to secure the victory.
Taurean Green attempted a desperation three-point shot at the buzzer, but Tennessee got the rebound.