Barry Bonds has equalled Hank Aaron's all-time record of 755 home runs, which has stood since 1974.
Bonds celebrates as he rounds third base after hitting his homer
The San Francisco Giants slugger hit the 382-foot homer off San Diego Padres pitcher Clay Hensley in the second inning at San Diego's Petco Park.
Bonds, 43, was widely cheered as he circled the bases and greeted by his team-mates at home plate, moments after being booed as he strode to the plate.
His achievement has been overshadowed by claims he has used steroids.
The left-handed Bonds has never tested positive for drug use, and denies having done so knowingly.
But rumours have persisted since his home run tally took off after he turned 35, moving past the legendary Babe Ruth into second place in May last year with his 715th career home run.
He equalled Aaron's record at a venue where a giant syringe was thrown on the field by a fan in protest last season.
I finally did something right. I hit it good enough to get out
"It's the toughest thing that I've ever done in my career," he said afterwards.
"It's literally different than any other milestone I've ever gone through. I can't explain the feeling of it, it's just Hank Aaron."
The left-handed hitter had gone 27 times to bat since he struck his last home run on 27 July in San Francisco.
But the wait ended with his first at-bat in Saturday's game, as he slammed the right-handed Hensley into the left-field stands.
"I was thinking I finally mechanically did something right," Bonds said. "I don't even think the pitch was a strike. It was up and away. I finally did something right, and I hit it.
"I didn't even know where that ball went. I knew I hit it, and I knew I hit it good enough to get out."
While some of the crowd still jeered as Bonds took his position in left-field for the bottom half of the second inning, most gave him a standing ovation and he responded by tipping and waving his cap to the stands.
Bonds turned around between pitches from his defensive post to take a long look at the spot where his home run had landed.
He was walked in his next three at-bats before leaving the game to another ovation in the top of the eighth.
"I want to especially thank the fans," Bonds added. "I just don't know what to say. I really appreciate the way San Diego handled it."
Bonds will not start the Giants' next game on Sunday, taking a day off to rest and reflect on his acheivement. "I'm going to celebrate with my family," he said.
He will start his attempt to break Aaron's record in a seven-game run at home, with four against the Washington Nationals starting on Monday, followed by three more against Pittsburgh, his former team.
Alex Rodriguez became the youngest player in Major League history to reach 500 home runs when he connected off Kyle Davies in the New York Yankees' 16-8 win over the Kansas City Royals on Saturday.
"A-Rod" reached the milestone in the first inning at 32 years and eight days, breaking the record of 32 years and 338 days held by Jimmie Foxx.
Rodriguez, who had gone 28 at-bats since hitting number 499 in Kansas on 25 July, was the 22nd player to join Major League Baseball's 500-homer club.
He connected in his 1,855th game, putting him third on that all-time list. Mark McGwire reached the milestone in 1,639 games and Babe Ruth in 1,740.