Assistant manager Matt Craddock has also left the club.
John said: “We thank you for the opportunity to work with Rovers Ladies and wish the team well for the future.”
John and Matt, who both have full-time coaching roles with The FA, took the reins in July following Scott Rogers’ departure to Liverpool last season.
They leave Rovers ninth in the FA Women’s Premier League Northern Division, having won just two of their eight league games.
Rovers’ Womens & Girls Development Officer, Gemma Donnelly, will take temporary charge of first team affairs until a permanent successor is appointed.
Further announcements are expected to be made in due course.
Gemma said: “We would like to thank John and Matt for their hard work and commitment over the past four months.”
Rovers get their Premier League Cup campaign underway at home to Sheffield FC this Sunday. Kick-off at the Anchor Ground in Darwen is at 2pm. All supporters are welcome.