The Italian football federation said in a statement the decision had come after the country's football finance watchdog deemed the clubs' balance sheets too precarious.
"Serie B side Ancona and 20 Lega Pro clubs will not be readmitted to their championships after the consideration of (football's financial watchdog) Covisoc's findings," the statement said.
Clubs complained that the decision, made in the wake of Italy's shock exit in the World Cup group stage, was too rash and could not be suddenly enforced in the middle of a transfer window.
"There's no turning back," federation president Giancarlo Abete told a news conference.
Abete also said a decison would be made at the next board meeting in August over a new head of the federation's technical committee, with former Italy great Roberto Baggio interested in the role having stayed out of the game since his 2004 retirement.
In a separate development, the Italian professional footballers' association has hinted at a possible threat to strike on the first day of the league season after a disagreement with the federation over several of the new rules.
"We are considering drastic industrial action," association chief Sergio Campana told reporters. The association threatened a similar strike for the first day of last season but the action was averted.
