The AC Milan striker finally missed from 12 yards in the second half of his side's 2-1 Serie A defeat to Napoli at San Siro
The Italy striker has developed a reputation as a deadly penalty-taker and up until the miss against Napoli he had successfully converted all 26 of his spot-kicks for club and country.
With Milan trailing 2-0 after 59 minutes, they were awarded a penalty after Balotelli himself had been brought down by defender Raul Albiol.
However, the controversial forward was denied from the spot when his effort to the goalkeeper's right saw Reina produce an excellent diving save to tip the ball wide of the post.
Determined to atone for his miss, Balotelli hit the crossbar after an impressive solo run before eventually netting a late consolation goal.
His eventful night was not over there, though, as he then received a red card for dissent after the full-time whistle had been blown.
Speaking after the match, Reina admitted he had been closely studying Balotelli's penalty-taking technique in the build-up to the game.
"As always, we studied the Milan players. We knew Mario was very strong at penalties, but I managed to get it," he said.
"It has been a great start to the season. We knew it was a long time since Napoli had beaten Milan at San Siro and getting a result like this against a strong side really boosts our confidence."