The brothers, who say they are innocent, are accused of having formed an organised criminal network in early 2021 in Romania, as well as in the US and Britain
The 47-year-old lefthander and the bagman he met at 1992 US Open sectional qualifying in Memphis parted ways as friends, each releasing a statement saying as much.
"After 25 very rewarding and memorable years, Bones and I have mutually decided to end our player-caddie relationship," Mickelson said. "Our decision is not based on a single incident. We just feel it's the right time for a change."
Mickelson has not won a title since the 2013 British Open, his fifth major crown after the 2005 PGA Championship and the 2004, 2006 and 2010 Masters.
Denying him a career Grand Slam is a US Open triumph and Mickelson withdrew from last week's edition of the event to attend his daughter's graduation.
Mickelson has finished as a US Open runner-up a record six times, including 1999, 2002, 2004, 2006, 2009 and 2013.
Mickelson's younger brother Tim, who filled in for a day when Mackay was sick at a World Golf Championships event in Mexico earlier this year, will take over as the caddie for "Lefty" for the remainder of the season starting with his next event, the PGA Greenbrier Classic on July 6-9.
"After an amazing 25-year run, Phil and I have mutually decided to go our separate ways," said Mackay, who this year has been recovering from a double knee replacement.
"Player-caddie relationships don't often last that long. I will always be grateful that I was around to witness so much of Phil's career."
Mickelson's success made Mackay's dream come true.
"When Phil hired me in 1992, I had one dream: to caddie in a Ryder Cup. Last year, at Hazeltine, Phil played in his 11th straight Ryder Cup. It was so cool to have a front-row seat," Mackay said.
"I wish Phil nothing but the best. His game is still at an elite level, and when he wins in the future (definitely the Masters), I will be among the first to congratulate him."
Mickelson showed he can still contend in majors, finishing second to Sweden's Henrik Stenson last July in a British Open thriller. Now he must do so without Mackay.
"My relationship and history with Bones far exceeds golf," Mickelson said, saying he looks to forward to "sharing life and friendship" forever.
Mickelson won his first PGA event in 1991 at Tucson, Arizona, as an amateur before meeting Mackay, the only one of his 42 career tour titles to come without "Bones" on his bag.
The brothers, who say they are innocent, are accused of having formed an organised criminal network in early 2021 in Romania, as well as in the US and Britain
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