The 55-year-old replaced John Sheridan in January and helped guide the club to National League safety last season.
But the new campaign has seen the Spireites struggle at the wrong end of the table.
Tuesday night’s 3-2 defeat at Altrincham kept the club third from bottom. It was their sixth defeat in eight matches, leaving the club in the relegation places.
Chesterfield will start their hunt for a new boss after Pemberton became the first manager in the division to leave his position this season.
Chairman Mike Goodwin said results this season had not met the board’s expectations.
“I had a meeting with John this morning and it was agreed that a change of manager would be in the best interests of the club,” he announced.
“John came in and did an impressive job during two spells as caretaker manager, prompting us to hand him the role on a permanent basis. Unfortunately, results have not gone as we all expected this season, leading to a change of manager being required.
“I would like to thank John for his hard work, honesty and integrity and wish him all the very best for the future.
“The search for a new manager is now underway and I have no doubt that there will be a number of strong candidates. We will carefully consider the applications and carry out a stringent interview process before making an appointment.”