The Catalan club confirmed on Friday night that Vilanova would not be able to continue in the role as he continues to receive treatment for cancer.
Vilanova, who replaced Pep Guardiola as coach of Barca last summer, had a tumour removed from his throat in late 2011, but suffered a relapse a year later. He underwent another operation on his parotid gland in December before beginning a course of chemotherapy and radiotherapy in New York.
Club president Sandro Rosell announced the news at a press conference, which was held with members of Barcelona's first team in attendance.
A successor will be appointed in the coming days.
"In next few days the vice-president Josep Maria Bartomeu and sporting director Andoni Zubizarreta will present the new coach, probably at the start of next week," Rosell said.
Rosell said the results of tests Vilanova underwent this week prompted his decision.
He said: "After evaluating the results of the routine follow-up tests, taken by Tito Vilanova this week, he has been presented the option of being able to undergo a treatment to continue managing his illness which will be incompatible with him being the head coach of the first team from now on.
"I want to ask everyone, especially the media, for him and his family, for the utmost respect for the privacy of all in this process which starts from today."
He added: "It's a tough blow but Barca have suffered many blows and we've got through them."
There was no official mention of a successor, but reports in Spain earlier suggested Joan Francesc Ferrer, known as Rubi, could take over from Vilanova in a caretaker capacity.
The 43-year-old joined Barca's coaching staff from second division Girona last month as an assistant to Vilanova and Jordi Roura - who took charge of the first team when Vilanova was absent last season.
In England, Tottenham boss Andre Villas-Boas was installed as the bookies' favourite, but the Portuguese was not keen to talk on the subject following the friendly against Colchester.
"It's a very difficult situation and all of my wishes are with Tito," he told the Daily Telegraph. "If he has to resign for a situation of illness it's not the moment to speak about any speculation whatsoever.
"It's not even a question, we are speaking about a person's health."
Rosell confirmed the club would not now be travelling to Poland for Saturday's pre-season friendly with Lechia Gdansk.
Vilanova, 44, was Guardiola's assistant before being promoted to the top job when the record-breaking coach decided to take a one-year sabbatical from the game last year after winning 14 trophies in four seasons.
In his one campaign in charge of the club, Vilanova led Barca to their fourth Primera Division title in five years, becoming only the second team after Real Madrid to reach the 100-point barrier.
They also reached the semi-finals of the Champions League and Copa del Rey, but lost to Bayern Munich and Madrid respectively.
- Sports & Recreation
- Pep Guardiola
- Tito Vilanova