The 14 Premier League clubs left out of plans for a European Super League have "unanimously and vigorously" voted against the proposals as England's top flight considers "all actions available" to halt the breakaway competition.
Manchester United, Liverpool, Arsenal, Chelsea, Manchester City and Tottenham have announced plans to join a lucrative new tournament.
The 'big six' suggested this would run alongside the Premier League, in place of the Champions League.
But their guaranteed involvement in the Super League has been widely criticised as anti-competitive, with Premier League performance having no impact on European fortunes.
The Premier League and The Football Association (FA) each condemned the idea when it was first reported on Sunday.
And the two bodies held a meeting for the 14 remaining Premier League clubs on Tuesday, at which the league added it would hold the 'big six' "to account under its rules".
"The Premier League, alongside The FA, met with clubs today to discuss the immediate implications of the Super League proposal," a statement read.
"The 14 clubs at the meeting unanimously and vigorously rejected the plans for the competition.
"The Premier League is considering all actions available to prevent it from progressing, as well as holding those shareholders involved to account under its rules.
"The league will continue to work with key stakeholders including fan groups, government, UEFA, The FA, EFL, PFA and LMA to protect the best interests of the game and call on those clubs involved in the proposed competition to cease their involvement immediately.
"The Premier League would like to thank fans and all stakeholders for the support they have shown this week on this significant issue.
"The reaction proves just how much our open pyramid and football community means to people."
This meeting took place as City manager Pep Guardiola announced his opposition to the Super League in a pre-match news conference.
Liverpool counterpart Jurgen Klopp said on Monday he did not support the plans but added the club's Boston-based owners Fenway Sports Group were "reasonable people" and "never have to explain these decisions to me or ask for permission".