The Italian football federation (FIGC) said on Monday that clubs who join a breakaway league would be banned from all domestic competitions including Serie A.
Three Serie A clubs — Juventus, Inter Milan and AC Milan — were among 12 major European sides who were involved in the plans to launch a European Super League last week.
"[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.
Inter Milan announced their withdrawal from the proposed Super League last Wednesday after the departure of the 'big six' English clubs.
But Juventus and AC Milan were less clear, acknowledging the failure of the plans without specifying whether or not they were leaving 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 rule will be added to the licenses of the clubs and into the regulatory texts of the FIGC.
The new regulation was adopted unanimously, including by the managing director of Inter Giuseppe Marotta, who sits on the FIGC federal council as a representative of the league, according to La Gazzetta dello sport.
The three Italian clubs have since continued to defend the idea of reform in European football.
"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.