NBA Executive Vice President Stu Jackson announced the $25,000 penalty on Monday.
House declined to comment on the fine as he prepared in the Heat's locker room for Monday's game against the Cleveland Cavaliers.
The league said the incident occurred with 22.2 seconds left in the game.
Though House has made the same gesture in the past without being reprimanded, Miami head coach Erik Spoelstra said it won't be an issue again.
"The league came down on [the gesture]," Spoelstra said, "so we've taken it and it won't happen again."
After hitting a game-sealing 3-pointer following an assist from LeBron James, House gestured beneath his crotch with his hands.
James weighed in on the league's decision Monday night.
"You never want anyone to be fined ... for the league to take money out of any of our pockets," James said. "We work hard for it. It's just respect for the game. We all respect the game, and we're going to continue to respect the game."
Information from The Associated Press was used in this report.