Rodriguez, who hit No. 599 on July 22 against Kansas City, went 51 plate appearances before hitting the milestone homer in the first inning off Shaun Marcum. The hit also snapped an 0-for-17 streak he brought into Wednesday's contest.
Rodriguez became the youngest player to reach 600 home runs. Hall of Famer Babe Ruth hit his at age 36, while Rodriguez turned 35 last week. He also became the second player to reach 600 home runs in Yankee pinstripes, joining Ruth.
Along with Rodriguez, Mark Teixeira and Derek Jeter powered the Yankees' offense. Jeter had his first four-hit game of the season, doubling twice, while Teixeira went 2-for-3 with three RBI in the victory, which snapped New York's three-game slide.
Phil Hughes (13-4) picked up the win after limiting Toronto to one run on four hits and two walks in 5 1/3 innings, while striking out five.
Marcum (10-5) lost for the first time since July 1 after giving up five runs on eight hits and a walk in six innings.
However, the home run storyline went to Rodriguez on Wednesday. He stepped to the plate in the first inning with Jeter on base and two away, and Marcum threw a 2-0 pitch away. Rodriguez belted it to dead center field and the crowd's cheers intensified on the home run's long way out.
It turned out to be the winning hit, thanks to Hughes and the Yankees' pitching staff. The right-hander retired the first six batters he faced before Overbay walked to begin the third. Edwin Encarnacion followed with a single, but Hughes got the next three hitters out to escape the situation.
Jeter and Teixeira doubled in the home half to make it a 3-0 game, and Hughes allowed his only run in the next inning. With two on and two away, Overbay doubled to right to plate Jose Bautista, who had hit a leadoff single. However, Hughes ended the rally by striking out Encarnacion.
After Hughes worked a 1-2-3 fifth, Marcum loaded the bases with nobody out in the bottom half. Teixeira hit a two-run single to left to extend the Yankees' lead to 5-1, but Marcum managed to get out of the inning.
Blue Jays catcher John Buck left the game in the fifth inning after taking a foul ball off his hand. He was replaced by Jose Molina...Rodriguez's homer came exactly three years after he hit the 500th of his career, which he got off Kyle Davies in the first inning of a game against the Royals. Rodriguez also hit his 100th career home run against Toronto, doing so on August 12, 1998.