Napoli beat Genk 4-0 at home on Wednesday (AEDT) to end a run of nine games without a victory in all competitions, securing second place in Group E behind Liverpool.
Rumours that Ancelotti was set to depart the Stadio San Paolo have been growing in Italy, with suggestions ex-Milan boss Gennaro Gattuso has already been lined up as his replacement.
After the Genk win, Ancelotti indicated he would not be resigning from his post but said he would be holding talks with club president Aurelio De Laurentiis over his future.
Napoli announced Ancelotti's departure in a short statement posted on social media just hours later.
The statement said: "Napoli has decided to revoke the role of technical manager of the first team from Mr Carlo Ancelotti.
"The relations of friendship, esteem and mutual respect between the company, its president Aurelio De Laurentiis and Carlo Ancelotti remain intact."