Lee has been with the club for 22 years, the last seven of which were as the Magpies' managing director.
Newcastle United was acquired by an investment group led by PIF, PCP Capital Partners and RB Sports & Media in October. The club's new Board will continue to oversee all operations.
The club has also begun a formal recruitment process to appoint a CEO and will make an announcement in due course.
Lee Charnley said: "To have been at Newcastle United for 22 years has been an honour and a privilege, and the club, its people and our city will always be very special to me.
"As it enters a new chapter in its history under new owners, I wish Amanda, the rest of the Board and everyone connected with the club every success."
Amanda Staveley said: "I would like to thank Lee sincerely for his support and professionalism during this time of change. His knowledge and assistance have been hugely appreciated by the club's new Board.
"Lee ends a long association with the club and on behalf of the Board, staff and players, I'd like to wish him the very best for the future."