Inter Milan, Juventus and AC Milan were the three Italian clubs to sign up for the ESL
The Italian Football Federation (FIGC) has approved a rule that would ban clubs who sign up to breakaway competitions from competing in Serie A.
It follows the collapse of the proposed European Super League which involved Juventus, Inter Milan and AC Milan.
The tournament sparked furious backlash but Juve, plus Barcelona and Real Madrid, are yet to abandon the project.
Clubs playing in leagues not recognised by Fifa or Uefa will now not be able to play in domestic tournaments in Italy.
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The FIGC added a new clause to its rules underlining that clubs registering for national leagues must agree they will not join private, unauthorised competitions.
Under the European Super League proposals announced on 18 April, clubs agreed to play in the new competition but wanted to continue to play in their domestic leagues.
The proposals were heavily criticised by fans, authorities, governments and players and eventually nine of the 12 clubs, including the Premier League's 'Big Six' and both Milan clubs, pulled out.
"At the moment we have no news about who has stayed and who is out of the Super League," said FIGC president Gabriele Gravina.
"If, by the deadline for applications to the national championships on 21 June, someone joins up to other private leagues, they will not take part in our championship."