Juventus have been told they face expulsion from Serie A if they remain part of the proposed European Super League.
The Serie A club, along with Barcelona and Real Madrid, are yet to withdraw from the ESL.
Italian Football Federation president Gabriele Gravina said: "The rules are clear.
"If Juventus is still part of the Super League when it enters next season, it can't participate in Serie A.
"I would be sorry for the fans but rules are rules and they apply to everyone."
Juve, Barca and Real continue to be committed to the project, and last week defended their involvement in the breakaway league in the face of what they call "threats" from Uefa.
But pressure is mounting on Turin club Juve to withdraw.
"We are all a bit tired of this tug-of-war between Uefa and these three clubs," said Gravina. "I hope this dispute can be resolved as soon as possible. I hope to be able to mediate between Juventus and Uefa.
"The rules foresee the non-participation in our championship if the principles established by the federation and Uefa are not accepted.
"It's not good for international football, Italian football, Juventus. We have already said that the football association respects the rules."
The ESL was announced on 18 April but within 48 hours the plans had fallen apart with the English clubs withdrawing after fan protests and UK government pressure.
The nine clubs who withdrew from the competition have been given a financial punishment by Uefa.
Manchester United, Manchester City, Liverpool, Chelsea, Tottenham and Arsenal, along with AC Milan, Inter Milan and Atletico Madrid, also committed to the European governing body and its competitions.
They have agreed to make a combined 15m euro (£13.4m) goodwill contribution to benefit children's and grassroots football across Europe.