Agnelli was summoned after wiretap evidence from an ongoing investigation into the 'Ndrangheta linked staff at the Serie A champions to the Calabria-based criminal network.
It follows claims by an Italian football federation (FIGC) official that Agnelli and three other club officials maintained “contact” with an alleged 'Ndrangheta member.
In a Twitter message last week Agnelli said he had “never met mafia bosses”.
And after receiving from federal prosecutors his summons to give evidence, Agnelli read out and then published a defiant statement of denial.
“As I said a few days ago, I have never met mafia bosses. At regular intervals, as you know, I meet with all different kinds of fans, whether they're ticket-holders or members of groups of ultras,” he told media on Saturday. “Ultra” groups are powerful and often have close ties with clubs.
“This is done in the full light of day and remains an integral part of a president's role at a football club.
“If some of these people are now regarded differently by the law, this is something which, at the time, was neither known by me or any staff members at Juventus.
“Some will argue these ultras and their leaders aren't whiter than white, and I agree. But in the eyes of the law, they were free men and for us were not subject to any kind of stadium restriction.”
Agnelli was forced on the defensive two weeks ago when FIGC official Giuseppe Pecoraro told the Anti-Mafia Commission the Juventus chief and three other club officials had “contacts” with alleged 'Ndrangheta member Rocco Dominello.
“Dominello has relations with the management of Juventus for the organization of tickets and season tickets,” Pecoraro told media on March 8th.
“For now, there have been no violations… because we're only talking about contacts between these people and staff at Juventus.”
Prosecutors are investigating the 'Ndrangheta's reach in the Piedmont region, whose capital is Turin, and Dominello is alleged to have had privileged relations with Juventus officials in connection with the acquisition and distribution of match and season tickets.
Agnelli defended Juventus staff, saying they have been victims of “bias”.
“This club, its employees and its members have nothing to hide or fear, that's why I am speaking today,” Agnelli added.
Juventus, often accused of benefiting from biased refereeing, are bidding for a record sixth consecutive Serie A title and a third consecutive league and Cup double.
They face Barcelona in the quarter-finals of the Champions League next month.