"You have to give our trainers Trainer of the Day for getting them back in there," manager Joe Girardi joked.
Jeter tied it with an RBI single in the seventh inning, Matsui put New York ahead with a solo homer in the eighth and Mariano Rivera worked a perfect ninth for his seventh save in eight chances.
"You know I don't like watching so it was good to get back out there," Jeter said. "It's been a couple of days. It's a big win for us. We needed that one."
Sabathia (3-3) yielded five hits in eight innings to win consecutive starts for the first time this season. He walked four, one intentional, and struck out five to help the Yankees take two of three from Toronto, the surprise leader in the AL East.
"I'm just trying to pound the strike zone, getting guys to swing early in the count, keeping the pitch count down and just trying to pick the team up," Sabathia said.
Sabathia, who pitched a four-hit shutout at Baltimore last Saturday, improved to 8-3 in 11 career games against the Blue Jays.
"He pitched well against one of the best offenses in the league," teammate Johnny Damon said.
Jeter and Matsui returned to the lineup after sitting out Wednesday night. Jeter missed two games with a sore right oblique and Matsui left Tuesday's game after one at-bat with a tight right hamstring.
Matsui led off the eighth with a drive to right-center off left-hander Jesse Carlson (0-2), his fourth.
"When he's swinging the bat well he can hit lefties or righties, it doesn't make a difference," Jeter said. "That was the difference in the game."
Jeter finished 1-for-5 with an RBI and Matsui went 1-for-3 with a walk.
New York went 4-2 on its road trip and returned to .500 at 17-17 as it prepares to begin a nine-game homestand. The Yankees host Minnesota for a four-game series beginning Friday night, with Baltimore and Philadelphia to follow.
Alex Rodriguez, who came off the disabled list in Baltimore last Friday, will get to play at the new Yankee Stadium for the first time.
• Blue Jays: Ryan activated, but closer role on hold
"All the guys have been telling me about our new home," Rodriguez said. "For me it's like opening day all over again."
Toronto lost a home series for the first time this season after winning four and splitting a two-game set with Cleveland.
Toronto took a 2-1 lead in the fifth. Scott Rolen drew a leadoff walk and scored one out later on Rod Barajas' double. John McDonald followed with a single but center fielder Brett Gardner threw out Barajas at the plate.
"That play at the plate was probably the biggest play of the game," Sabathia said.
New York tied it in the seventh. Gardner led off with a walk against Brian Tallet and was running when Francisco Cervelli singled past shortstop Marco Scutaro, who was moving over to cover second. The Blue Jays brought in right-hander Jason Frasor, but Jeter drove in Gardner with a bloop single to right.
Tallet was disappointed in himself for issuing the leadoff walk to Gardner.
"I got the guy out two times, what am I doing walking him?" he said. "In that situation you've got to make [him] hit the ball and let your defense play behind you. I didn't and the end result ended up being a run."
Toronto loaded the bases against Sabathia with two outs in the seventh but Aaron Hill grounded out.
Hill started at designated hitter after fouling a ball of his shin and leaving after five innings Wednesday. McDonald played second base in his first start of the season.
Tallet allowed two runs and four hits in six-plus innings. He walked a season-high five.
"[Tallet] gives up a couple of runs against Sabathia and you figure you might have a chance to win that game," Blue Jays manager Cito Gaston said. "But we didn't. We move on."
- Toronto activated LHP B.J. Ryan off the 15-day disabled list after the game and optioned LHP Bill Murphy to Triple-A Las Vegas. Ryan has been out since April 23 with muscle tightness in his back and shoulder. LHP Scott Downs will remain Toronto's closer.
- New York RHP Chien-Ming Wang (hip) will make a second rehab start with Triple-A Scranton/Wilkes-Barre on Sunday.
- Buffalo Bills quarterback Trent Edwards and receiver Lee Evans took batting practice before the game. Edwards hit two home runs during his session and later threw out the first pitch. Evans handled PA duties during the bottom of the third.
- A male fan seated in the front row behind the third-base dugout was cut on the back of the head by Toronto slugger Vernon Wells' broken bat in the fourth. He walked away to receive medical attention and later returned to his seat.