Cardinal Bertone will host a seminar to rebut the book's claims
Cardinal Tarcisio Bertone, Archbishop of Genoa, broke the church's official silence on the controversial book.
Its story about the Church suppressing the "truth" that Jesus had a child with Mary Magdalene has convinced many fans.
But the cardinal's spokesman denied reports that the clergyman was asked by the Vatican to hit back at the book.
Carlo Arcolao told the BBC's News website that it had been the cardinal's own decision to make a public statement about the book.
Mr Arcolao confirmed that the cardinal told an Italian newspaper: "It astonishes and worries me that so many people believe these lies."
The archbishop told Il Giornale: "The book is everywhere. There is a very real risk that many people who read it will believe that the fables it contains are true."
The Da Vinci Code, by US author Dan Brown, has been a publishing sensation around the world and is still in best-seller lists.
Its conspiracy theories and thriller style, in which two code-breakers try to track down the truth behind the Holy Grail, have caught the imaginations of millions.
Its central claim is that the Holy Grail is really the bloodline descended from Jesus and Mary Magdalene - which the Church is supposed to have covered up, along with the female role in Christianity.
Brown has previously said: "All of the art, architecture, secret rituals, secret societies, all of that is historical fact."
On Wednesday, Cardinal Bertone will host a seminar called Storia Senza Storia (Story Without History) to rebut the claims.
The cardinal has been deputy to Cardinal Joseph Ratzinger, the man known as "the Pope's enforcer" and a possible successor to the pontiff.
As well as the original novel, published in 2003, another 10 books have been written to debunk its claims and a booming tourist industry has sprung up around its sites.It is also being turned into a film starring Tom Hanks, Jean Reno and Audrey Tautou. Publisher Transworld was unavailable for comment.