The Serie A side made the decision just hours after Ancelotti presided over a 4-0 victory over Genk in the Champions League, which ensured they qualified for the Round of 16.
“Napoli have decided to relieve Ancelotti of his duty as a manager,” the club said in a statement published late on Tuesday night.
“The current relationship between Ancelotti, the president Aurelio De Laurentiis and the club – which is based on friendship, mutual esteem and respect – remains unchanged.”
Immediately after the victory over Genk, Ancelotti had told reporters he had no intention of resigning from his post.
“I’ll see the President [on Wednesday] and we’ll see,” he said.
Ancelotti, who previously managed Chelsea, has been linked to Premier League sides Arsenal and Everton in recent weeks.