Clubs that enter a breakaway league will be barred from all domestic competitions, including Serie A, according to the Italian soccer federation (FIGC).
Last week, 12 big European clubs, including Juventus, Inter Milan, and AC Milan, were interested in the preparations to create a European Super League.
“(Clubs) who plan to play in competitions outside of FIFA or UEFA will lose their affiliation to our championship,” FIGC President Gabriele Gravina said after a meeting on Monday.
Following the departure of the "big six" English clubs, Inter Milan announced their withdrawal from the planned Super League on Wednesday.
Juventus and AC Milan, on the other hand, were less forthcoming, admitting the plans' failure but refusing to say whether they would abandon the project.
“It is obvious that if on June 21, the closing date for applications for registration, some wanted to participate in competitions of a private nature, they would not take part in our championship,” said Gravina in a statement.
“At the moment, we have no information on who stayed and who left the Super League.”
The new regulation will be incorporated into club licenses as well as FIGC regulatory texts.
According to La Gazzetta dello Sport, the new regulation was unanimously approved, including by Inter's managing director, Giuseppe Marotta, who sits on the FIGC federal council as a representative of the league.
Since then, the three Italian clubs have started to advocate for European football reform.
“Football is no longer a game but an industrial sector and it needs stability,” argued Juventus club president Andrea Agnelli in Italian newspaper La Repubblica last Wednesday.