The 89-year-old president confirmed his availability as a witness in a letter to the court, La Repubblica reported.
He will testifiy at 10am on October 28th from the Quirinal Palace in Rome, the president's official residence.
The case dates back to the early 1990s when Nicola Mancino, a former interior minister, allegedly negotiated with the mafia to stop a string of bomb attacks in Sicily that killed 21 people, including the anti-mafia judges Giovanni Falcone and Paolo Borsellino.
Cosa Nostra, the Sicilian mafia, is said to have offered to stop the attacks in return for lighter jail terms and conditions for convicted gang members.
Mancino is accused of pergury but has denied any wrongdoing. Others on trial include mafia bosses Totò Riina, Leoluca Bagarella and Bernardo Provenzano, as well as Marcello Dell'Utri, a former politician who is also in jail over mafia links.
Riina and Bagarella have also requested to participate at the president's hearing, La Repubblica reported.
Napolitano became embroiled in the case when prosecutors wiretapped Mancino's phone and recorded four calls made to the president between November 2011 and May 2012, which appeared to be seeking his help.
But in May last year the Palermo court ruled that he could not be asked about the information contained in the recordings, which were destroyed after Napolitano petitioned to the Constitutional Court that prosecutors had gone beyond their powers in keeping them, Ansa reported.
Instead he will be questioned about a letter he received from a legal advisor, Loris D'Ambrosio, in July 2012.
In the letter D'Ambrosio, who died shortly after writing it, reportedly told the president about his fears that there may have been talks between the government and the mafia in the early 1990s.