The Catalan giants confirmed on Friday that the 44-year-old would not be able to continue in his role as he continues to receive cancer treatment.
In an open letter published on the club's official website, Vilanova wrote: "After five wonderful years as part of a team that has made dreams come true for any coach, it's time to face a change in my professional life and focus my energy on continuing to fight the disease I was diagnosed with a year and a half ago.
"The doctors recommend that, with the treatments that I must follow from now on, I cannot devote 100 per cent to the tasks of head coach as required by FC Barcelona. But I will continue to work closely for the club that I love on other sporting tasks."
Vilanova was Pep Guardiola's assistant before being promoted to the top job last summer when the record-breaking coach decided to take a one-year sabbatical from the game 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 mark.
He acknowledged it was difficult to stand down from his position, saying: "It is not easy to leave this very special group of players, fellow staff and friends with whom I shared many memorable experiences. I'll be eternally grateful for everything you have given me and the affection you have shown."
Vilanova 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, where Guardiola was taking a year-long sabbatical after leaving the Nou Camp hot-seat.
Vilanova, who earlier this week claimed Guardiola "wasn't there for me" during his rehabilitation, went on in his letter to thank club officials and doctors for their support.
"I also want to especially thank the president (Sandro Rosell), the board and the sporting director Andoni Zubizarreta for the confidence they put in me and, above all, the unconditional support, both sporting and personal," he wrote.
"Equally important to me is the support I receive - both medical and humane - from Dr Ramon Canal and his team of doctors who are on my side. I do not play this game alone, but as part of a supportive club that will help me succeed in this long process, as it has done until now."
He added: "To all of you, members and fans of the club, I also want to thank you wholeheartedly for the support and affection that you have shown - not just now but over the last few months. I can tell you I'm calm, strong and that I start this new stage in my recovery with full confidence that all will be well."
Rosell said that the club expect to make an announcement on Vilanova's replacement in the coming days, 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.
Vilanova ended his letter by requesting "tranquility and privacy", and wishing "much luck and every success to the new coach of the best team in the world".
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