The 44-year-old will step down with immediate effect following just two-and-a-half months in charge of the team.
A statement on the club's official website read: "The board of Cheltenham Town FC have reached an agreement with manager Paul Buckle for him to leave the club. Neither party will be making any further statement on this issue."
Head of academy coaching Russell Milton will take charge of proceedings for Saturday's home match against Bury.
Cheltenham are currently third from bottom in the league table after taking 30 points from as many games.