Dallas Cowboys owner Jerry Jones has warned that any of his players who disrespect the US flag will not play.
Mr Jones said the NFL, the US's most prestigious American football league, cannot leave the impression it tolerates such behaviour.
It follows a feud between US president Donald Trump and players who have staged the so-called "taking a knee" protest during the national anthem.
Mr Jones had his strongest comments so far on the national anthem controversy on Sunday night.
They started with his response to a question about Vice President Mike Pence leaving the game in Indianapolis after about a dozen San Francisco players knelt during the anthem.
"I know this, we cannot ... in the NFL in any way give the implication that we tolerate disrespecting the flag," said Mr Jones, also the team's general manager, after a 35-31 loss to Green Bay.
"We know that there is a serious debate in this country about those issues, but there is no question in my mind that the National Football League and the Dallas Cowboys are going to stand up for the flag. So we're clear."
The Cowboys and Mr Jones knelt arm-in-arm before the anthem when they played at Arizona two weeks ago, a few days after Mr Trump criticised NFL players for anthem protests.
All of them stood during the anthem, with arms still locked.
Mostly Dallas players have stood on the sideline, many with hands over their hearts, during the anthem since former 49ers quarterback Colin Kaepernick started kneeling last season in protest of police treatment of African-Americans.
Mr Jones said he was not aware of whether any of his players had raised a fist at the end of the anthem before the Green Bay game.
"I don't know about that," said Mr Jones, who was inducted into the Pro Football Hall of Fame in August.
"But if there's anything that is disrespectful to the flag, then we will not play. OK? Understand? If we are disrespecting the flag, then we won't play. Period."
The 74-year-old Mr Jones said showing respect for the flag and the anthem is more important to him than any potential issues of team unity.
"There is no room here if it comes between looking non-supportive of our players and of each other or creating the impression that you're disrespecting the flag, we will be non-supportive of each other," Mr Jones said.
"We will not disrespect the flag."
Mr Jones said a phone conversation with Mr Trump after the display in Arizona included Mr Trump telling him there was a rule on the books.
The NFL has said the game operations manual distributed to teams includes a reference to players standing for the anthem, but that it is a policy and not a rule.
The league has said it does not plan to punish players over anthem protests.
"The league in mind should absolutely take the rules we've got on the books and make sure that we do not give the perception that we're disrespecting the flag," Mr Jones said.