"That was pretty cool," Rays manager Joe Maddon said. "That ball was properly struck right there."
Kyle Farnsworth (2-0) got the final out in the top of the ninth for the win. Tampa Bay has won seven of nine.
Reyes dropped to 0-11 in 24 starts since a win against the Angels on June 13, 2008, while with Atlanta. He allowed one run and four hits in six innings Tuesday.
"Overall, he did much better than the outcome will suggest," Toronto manager John Farrell said. "He threw the ball very good."
The Blue Jays were without slugger Jose Bautista, who didn't play because of neck spasms. Farrell said Bautista might be out until this weekend.
Bautista underwent an MRI exam that showed no structural issues.
"The MRI just shows the spasms," Farrell said. "There's no spine issues or anything like that. He'll have to go through some baseball activity once he's symptom-free."
Tampa Bay All-Star third baseman Evan Longoria was activated from the 15-day disabled list after missing 26 games due to a left oblique strain and had a single in four at-bats. He struck out twice.
Rays starter Wade Davis had his three-game winning streak end. The right-hander gave up two runs and six hits in six innings.
Toronto went ahead 2-1 in the fourth inning on a run-scoring double by Edwin Encarnacion. The Blue Jays loaded the bases with two outs later in the inning, but failed to extend their lead when Rajai Davis struck out.
Adam Lind put the Blue Jays up 1-0 with an RBI single in the first, giving him 14 RBIs over his last 11 games.
Kelly Shoppach, hitless in his 17 previous at-bats, tied it at 1 on a second-inning RBI bloop single.
Tampa Bay rookie Brandon Gomes, recalled from Triple-A Durham, walked one over two scoreless innings in his major league debut.
"He looked great," Davis said. "I know he was nervous. Poise was really good."
Rays DH Johnny Damon was hit on the underside of the left forearm by Reyes' pitch in the third, but remained in the game. He missed three games from April 18-20 after breaking the tip of his left ring finger while trying to bunt.
Three of Upton's four career walkoff hits have come against Toronto. ... Maddon tweaked his rotation and will have LHP David Price pitch in the series finale Thursday. Price was first scheduled to pitch this weekend at Baltimore. ... Blue Jays INF Aaron Hill (strained right hamstring) is set to play in an extended spring training game on Wednesday. He could be ready to return early next week. ... Tampa Bay 2B Sean Rodriguez (dislocated left pinky finger) was back in the lineup. ... Former Tampa Bay closer Roberto Hernandez will be working with Rays relievers during homestands. ... The Rays lead the all-time series 113-112.