Barcelona president Sandro Rosell confirmed at a press conference that Vilanova would not be continuing in the role as he requires further treatment for the throat cancer which was first diagnosed in late 2011.
Vilanova, who replaced Pep Guardiola as coach last summer, suffered a relapse last year and underwent a second operation in December before beginning a course of chemotherapy and radiotherapy in New York which saw him miss a number of matches in the second half of the 2012/13 season.
The 44-year-old returned to the bench to lead Barcelona to the Primera Division title - becoming only the second team in La Liga history after Real Madrid to reach 100 points in the process - but it has now been confirmed he will not be returning for a second season in charge of the Catalan giants as he continues his fight against the illness.
Famous names from the world of sport were keen to wish Vilanova the best in his battle following the announcement, including Barca superstar Messi.
He wrote on his Facebook page: "Strength Tito! We are all with you in this fight!"
The Argentinian's club-mate Andres Iniesta added in AS: ""Tito will once again win this terrible battle facing him. On behalf of the players and everyone, I send him all my love and support. At the end of the day, this is about a person, not a coach."
Midfielder Thiago Alcantara, who this week left Barcelona to join former coach Guardiola at Bayern Munich, added on his Twitter account: "All the encouragement in the world for Tito and his family. We are all with you!!"
Liverpool's former Barcelona goalkeeper Jose Reina added: "Football is equally fleeting in its victories and defeats. All my support for Tito Vilanova and his family! Support! You are going to win this game!"
Rival clubs both in Spain and abroad also came out in support of Vilanova, including Real Madrid who released a statement on their website which read: "Real Madrid CF want to express their support for and solidarity with Tito Vilanova and also to his family, friends and FC Barcelona."
Chelsea, whose new coach Jose Mourinho was in charge of Madrid last season as they battled Barca for the Primera Division title, Tweeted: "Everyone at Chelsea Football Club sends their best wishes to Tito Vilanova for a speedy recovery.
"Jose Mourinho, his coaching staff and the Chelsea players also all send their best wishes to Tito Vilanova for his fast recovery.
The support for Vilanova was not limited to the world of football either.
Spanish cyclist Alberto Contador, the two-time Tour de France winner, wrote on his Twitter account: "From the Tour I'm sending lots of encouragement and strength to Tito Vilanova like all those fight against this damn illness."
NBA star Pau Gasol, who was born in Barcelona and played for his home-town club before moving to the United states, added: "The first and most important thing in life is health. My best wishes to Tito Vilanova in this difficult moment. #animsTito #GetWellSoon".
Barca are set to name Vilanova's successor next week, with the likes of Marcelo Bielsa, Gerardo Martino, Andre Villas-Boas, Michael Laudrup, Luis Enrique and Rubi, who recently joined the Catalan club's coaching staff, being linked with the post in the Spanish media.