"Our decision is not based on a single incident. We just feel it's the right time for a change," Mickelson, 47, said in a statement.
"My relationship and history with Bones far exceeds golf. He has been one of the most important and special people in my life since the day we met and I will always be grateful for everything he has done for me."
The former world number two said his caddie for the rest of the year will be his brother Tim, who last July stepped down as head of Arizona State's men's golf programme to become Spaniard Jon Rahm's agent.
Mickelson's last event with Mackay was the St. Jude Classic earlier in June where he finished in ninth place.
He withdrew from last week's U.S. Open in Wisconsin so he could attend his daughter's graduation in California but still had Mackay examine the course in the rare event he would be able to make his tee time.
"After an amazing 25-year run, Phil and I have mutually decided to go our separate ways," said Mackay.
"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."
(Reporting by Frank Pingue in Toronto; Editing by Toby Davis)
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