Club president Sandro Rosell told a hurriedly called news conference at Barcelona's training facility on Friday that Vilanova will "follow a treatment" that will be "incompatible" with staying on as coach.
Rosell said that a new head coach would be named next week and that Barcelona was cancelling its first friendly of the preseason, scheduled to be played in Poland on Saturday against Lechia Gdansk.
Spanish media was rife with speculation that little-known Joan "Rubi" Ferrer would replace Vilanova. Ferrer was hired on June 28 as Barcelona's new assistant coach after his strong season with Catalan club Girona in the second division.
In Vilanova's single season in charge he led Barcelona to the Spanish league title with a record-tying point 100 points. In doing so, he played his part in denying Real Madrid a major trophy and the exit of counterpart Jose Mourinho, the man who originally made Vilanova famous for poking him in the eye during a scuffle between the two heated rivals.
The 44-year-old Vilanova had twice previously been obliged to take a medical break from his duties at Barcelona, once as Pep Guardiola's assistant in the 2011-12 season and again as its manager last season.
The quiet-spoken coach had battled the recurrent tumour in a saliva gland and had returned to work on both occasions.
As recently as April, Vilanova said that he felt fine and "had never thought about quitting" the job of coach that he called "his life."
But at that point his doctors had encouraged him to continue working. Now, that has apparently changed.
"This is a piece of news I would have never in my life wanted to give," Rosell told a packed media room including Barcelona's first team with Messi and captain Carles Puyol.
"After evaluating the results of the routine tests and checkups for Tito Vilanova, the option arose of following a treatment that will be incompatible with the development of the responsibilities as the coach of the first team," Rosell said.
Vilanova was not in attendance.
He first fell ill in Nov. 2011 and had to have surgery. He then had a second tumour removed last December and travelled to New York several times to receive further treatment.
In his absence last season, assistant Jordi Roura took over to mixed results in the Champions League before Bayern Munich crushed Barcelona 7-0 on aggregate in the semifinals — with Vilanova back on the touchline.
First as Guardiola's assistant, Vilanova provided the tactical know-how that helped his boss forge one of the best teams in the history of the sport — winning 14 of a possible 19 titles from 2008-2012, including two Champions League trophies.
As a young player, Vilanova spent six years at Barcelona's training academy — from 1984-90 — but never made it to the topflight team, going on to play for clubs such as Celta Vigo and Mallorca before his playing career was cut short by a serious knee injury. That injury led Vilanova into coaching and to Barcelona's cadets, tutoring current first-team players Messi, Cesc Fabregas and Gerard Pique.
Vilanova moved on from Barcelona to work as sport director at third division club Terrassa before answering Guardiola's call to help him take Barca B up from the third division in 2007-08.
Barcelona is set to play Bayern, Guardiola's new club, in a friendly on Wednesday.
"This is a very hard blow, but we have bounced back from others," Rosell said. "You must think about the individuals first and ... the club second."
Associated Press writer Harold Heckle in Madrid contributed to this report.