The former Barnsley boss was dismissed following a run of just six wins in 26 league games this season.
Wilson, whose final game in charge was a 2-0 defeat by Bradford City on Saturday, leaves Chesterfield in the relegation zone on goal difference.
"We feel that a change to the management team is necessary at this time to give us the best possible chance of securing our League One status," said Chesterfield director and company secretary Ashley Carson in a statement.
"We hope to be able to announce a new management team soon."
Left-back Ritchie Humphreys has been named as Chesterfield caretaker manager in the interim.