The manager has left the ProAct as the Spireites continue to languish in the bottom four.
Chesterfield had high hopes this season but just one win since October has the club struggling at the wrong end.
John Pemberton has been named as caretaker manager and will take the helm for Saturday’s visit of in-form Sutton United.
Second from bottom Chesterfield will begin their search for a new boss immediately.
Company secretary Ashley Carson said: "The decision was taken after full discussion last night with majority shareholder Dave Allen and also with Mike Goodwin, chairman of the Community Trust, who is heading up the potential takeover of the club.
"Whilst I think we all knew the time was right to make this change, it is important that I work very closely with Mike Goodwin, as ultimately we look forward to a change of ownership very soon.
"There will be a joint plan going forward so that any decisions taken regarding the appointment of a new manager will be made in full agreement with the Community Trust."