Sheridan had been on gardening leave since August 28 after the Spireites failed to win any of their first four league and cup matches this season. Assistant manager Tommy Wright and coach Mark Crossley have been in temporary charge of first-team affairs since.
Sheridan's exit has now been finalised, with the League Two club saying in a statement: "John Sheridan has resigned from his position as manager of Chesterfield FC with effect from September 18.
"The club wishes to record its appreciation of Mr Sheridan's achievements in leading the club to promotion from League Two to League One in the 2010/11 season and winning the Johnstone's Paint Trophy last term. The club wishes him well for the future."
Sheridan led Chesterfield up into League One with a title-winning 86 points in 2011, an achievement which saw him named League Two Manager of the Year by the League Managers' Association.
He guided the Spireites to Johnstone's Paint Trophy glory at Wembley last season - the first time the club had won the competition in their history. However, they were not able to avoid relegation from League One, losing six of their final 10 games as they finished the campaign eight points from safety.
