"Cristian stepped in at a difficult point in our season and I want to thank him for the professional manner in which he and his coaching staff have conducted themselves during such a challenging time," the club's chairman Daniel Levy said in a statement.
Assistant coach Ryan Mason will take over head coach duties with immediate effect, the statement said.
Stellini had replaced fellow Italian Antonio Conte last month with Spurs still in fourth place. The defeat at St James' Park leaves them six points behind Newcastle and Manchester United having played more games.
Mason will take over for the remainder of the season while the club continues to look for a permanent manager alongside their search for a director of football following Fabio Paratici's resignation this month.
It marks the second time Mason has taken over as interim coach having taken the reins when the club parted ways with Jose Mourinho in April 2021.
Stellini was a member of Conte's managerial staff and the Italian had stepped in to lead the team when Conte missed a number of games due to gallbladder surgery.
In Conte's absence, Stellini guided Spurs to wins over Manchester City, West Ham United and Chelsea.
However, he was unable to recreate that success after replacing Conte, with Spurs winning only one game against Brighton & Hove Albion while Bournemouth snatched a last-gasp 3-2 away victory earlier this month.
