Vilanova was diagnosed with throat cancer for a second time in December 2012 and was forced to resign at the end of his lone season in charge of Barcelona, which resulted in the team winning La Liga.
In recent days it was reported that Vilanova's condition had worsened, and the club announced on Friday that its former coach had lost his battle with the disease.
A statement from Barcelona read: "FC Barcelona regrets to report that Francesc 'Tito' Vilanova has left us today, at the age of 45 years.
"The club wishes to express deepest condolences to his family, who shared those moments of pain along with partners, FC Barcelona fans and all of football and sport."
Prior to taking charge of Barcelona for the 2012-13 season, Vilanova served as an assistant to Pep Guardiola for four successful campaigns, which netted the club three league titles, two Champions League trophies and two Copa del Rey crowns.
As a player, Vilanova came up through the youth ranks at Barcelona as a midfielder but never made a first-team appearance, instead featuring for Spanish sides Celta Vigo, Mallorca and Elche among others.