The Cincinnati Reds outfielder homered off Florida lefty Mark Hendrickson in the first inning. Griffey joined Barry Bonds, Hank Aaron, Babe Ruth, Willie Mays and Sammy Sosa.
The 38-year-old homered with Jerry Hairston on third and one out. The left-handed swinger launched a 3-1 pitch 413 feet into the right-field seats.
Griffey received a standing ovation from the relatively sparse crowd and responded by coming out of the Reds dugout and tipping his helmet to the fans.
The game was the last one of an eight-game road trip for the Reds, who will return home Tuesday night to play the St. Louis Cardinals.
Controversy ensued in the stands following the home run. Justin Kimball, a 25-year-old from Miami, said he caught the home run ball, put it in a wool cap and then had the cap ripped from his hands. Kimball said someone ran off with the ball.
Police said they had found the fan with the baseball and would look at video tape to see if Kimball's claims could be supported.
However, the Florida Marlins announced Major League Baseball had authenticated the home run ball for a middle-aged male fan who would only give his first name as Joe.
The slugger hasn't enjoyed many golden moments since the Reds got him from Seattle in 2000. This will rank as one of his best with Cincinnati and, possibly, one of his last, given that he's in the final year of his contract.
It was a long time coming.
Griffey, one of baseball's most prolific sluggers before injuries began to take their toll, started the season with 593 home runs.
It took 216 at-bats to make history - his previous homer came May 31.
Griffey hit No. 597 on April 23 at Great American Ball Park, then went 90 at-bats - the second-longest drought of his career - before connecting again in San Diego on May 22.
He went another 29 at-bats, and even got a day off during the week to work on his swing, before hitting No. 599. Griffey went 17 at-bats between that homer and No. 600.
Like his 400th and 500th, this home run came on the road.
Unlike Bonds and Sosa, Griffey has stayed clear of questions about whether he came by all of his homers legitimately. His name has never come up in baseball's steroids scandal. Unlike Sosa, he's never been caught using a doctored bat.
Although Junior is linked numerically with Hammerin' Hank and the Babe, he has never been defined by the home run.
His game is so well-rounded that he was voted an All-Century outfielder with Seattle before his 30th birthday. By then, his backward cap and light-up smile were the face of baseball.
His statistics were setting the pace, too. When Griffey was traded to his hometown team before the 2000 season, he was significantly ahead of Aaron's record home run pace.
It seemed like a sure bet that when his nine-year, $116.5 million contract was wrapping up this year, he'd be the next home run king, or close to it. Then, the city would have two of its own atop baseball's revered lists - Pete Rose as the hits king, Junior as the home run king.
It hasn't turned out that way.
Griffey hit 40 homers in his first season with the Reds, when he became the youngest to reach 400 career. Then came a succession of major injuries - torn hamstrings, torn patella tendon, separated shoulder, torn ankle - that knocked him way off Aaron's pace.
Nearly knocked him off the map, too.
The one-time superstar got booed in his hometown and overlooked in conversation about the game's best players. It took him more than four years to get to homer No. 500 in 2004.
It seemed he might never make it to 600.
A year later, he was back in the swing.
Griffey hit 35 homers in 2005, winning the comeback player award. He followed it with 27 homers in 2006.
Last season, he played in 144 games - his most since 2000 - and hit 30 homers, leaving him seven shy of No. 600. The Reds erected a countdown board at Great American Ball Park, and featured him on the cover of the 2008 media guide.
While he closed in on the prominent power number, Griffey gave it little thought. He's never spent much time thinking about his statistics. He preferred to wait and talk about No. 600 when he got it.
Until then, his personal homer list would have to speak for itself.
Griffey was the youngest player in the majors - still only 19 - on April 10, 1989, when he homered off the Chicago White Sox's Eric King on the first pitch he saw at Seattle's Kingdome.
Homer No. 36 was one of his most satisfying. It came one batter after his father, Ken Sr., homered off California's Kirk McCaskill on Sept. 14, 1990, an unprecedented father-and-son moment in the majors.
Even now, Griffey says those two seasons he spent playing with his father in Seattle were the best times of his career. And he has suggested that he would like to finish his career back there.