Leeds United, Hull City and Sheffield Wednesday's Championship rivals part company with manager
The 49-year-old has left after a mutual agreement to end his tenure was struck.
His last game in charge was before the international break, when the Lions drew 2-2 with Hull City.
He leaves with Millwall sat 15th in the Championship table, three points adrift of the play-offs.
Millwall chairman James Berylson said: “We greatly appreciate Gary’s efforts throughout his time in charge and he departs with the best wishes of everybody connected with the club.
“While my time working directly with Gary has been short, I have thoroughly enjoyed all our interactions and feel that he leaves us having enhanced the club’s on-pitch status during four successful years in charge.”
Rowett took charge of the club in 2019, overseeing a run of mid-table finishes in the second tier.
He said: “Firstly, I would like to thank the fans for all their support during my time at the club. The atmosphere they create is truly unique.
“I have thoroughly enjoyed being manager of Millwall and depart with a feeling that the club has progressed during my tenure. As is sometimes the case in football, the time just feels right to part ways.
“I would like to thank John Berylson, James, Steve Kavanagh, Alex Aldridge, my backroom staff, and everyone behind-the-scenes for their hard work and support.
“Finally, I’d like to extend my gratitude to the players, who I thank for the blood, sweat, and tears they have put into their efforts. It has been a successful and enjoyable four years.”
Adam Barrett will take the reins on a temporary basis, supported by fellow coach Paul Robinson.