The National Football League will fine teams if players on the field refuse to stand for the national anthem, the league said.
Some NFL players knelt during the anthem to protest police shootings of unarmed black men, sparking a controversy that pulled in even US President Donald Trump, who criticised the players as being unpatriotic.
NFL Commissioner Roger Goodell on Wednesday said teams will be fined if players on the field fail to stand during the Star-Spangled Banner.
Players who choose not to stand may now remain in the locker room until after the anthem is finished.
The NFL Players Association criticised the new policy, saying it was not consulted and that it may issue a challenge.
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The policy rekindled the debate about whether kneeling is an affront to America or a patriotic expression of free speech, and has again pitted billionaire owners against mostly African-American players.
"Telling peaceful protesters to leave and do it behind closed doors is dangerous and un-American," the American Civil Liberties Union said on Twitter.
The White House did not immediately respond to requests for comment on Wednesday, but Vice President Mike Pence tweeted a headline about the new policy with the comment "#Winning."
However, there were some signs of dissension among team owners. San Francisco 49ers owner Jed York said his team abstained from the league vote on the policy, The New York Times reported, and New York Jets chairman Christopher Johnson told Newsday he would pay any fines.
"If somebody (kneels) takes a knee, that fine will be borne by the organisation, by me, not the players," Johnson said.
Former San Francisco 49ers quarterback Colin Kaepernick started the protests in 2016, and was subsequently shunned by all 32 teams in the league, going unsigned for the entire 2017 season.
Players and commentators have questioned whether he was being blackballed by the owners.
He has filed a grievance against the league, saying the owners colluded to keep him from being signed.
