Landon Donovan said his San Diego Loyal "would not stand" for bigotry having left the pitch in protest after an alleged homophobic comment was aimed at midfielder Collin Martin, who is gay.
An opposition player from Phoenix Rising was accused of making the comment just before half-time in their second-tier USL Championship game.
Loyal players returned after the break but walked off when the referee blew his whistle, forfeiting a match they were leading 3-1 and ending their chances of reaching the play-offs.
They forfeited a match against LA Galaxy II last week after an alleged racial slur was aimed at midfielder Elijah Martin. The player accused of making the comment has since been let go by Galaxy.
Loyal players took a knee before walking off during Wednesday's second division game against Phoenix Rising.
Loyal manager and executive vice-president Donovan, who played in the Premier League for Everton, said: "We went through a really hard incident last week in the LA match and we made a vow to ourselves, to our community, to our players, to the club, to USL, that we would not stand for bigotry, homophobic slurs, things that don't belong in our game."
Martin had been sent off after the incident but the red card was rescinded.
Instead, one was given to 38-year-old former forward Donovan - the United States' second-most capped player - after he came on to the pitch in the aftermath.
Phoenix Rising said the player who was alleged to have made the homophobic comment "vehemently denied" the allegations.
"I lost it because I know what this team has gone through," said Donovan. "I know how hard it was for them to even take the field tonight given everything that happened, and then for it to happen again a week later was just devastating for me."
Donovan said, despite players wanting to continue, the team decided that if the opponent who was alleged to have made the homophobic comment was not removed by either the referee, or by his club, they would not play.
"If they're not willing to act, then we have to act," he added.
"Our guys, to their immense credit, said we were not going to stand for this. They were very clear in that moment that they were giving up all hopes of making the play-offs, even though they were beating one of the best teams in the league.
"They said it doesn't matter. There are things more important in life and we have to stick up for what we believe in.
"I have tremendous pride in this group and this organisation."
In a statement, the USL Championship said it was aware of the allegation and an investigation was under way.
"Foul and abusive language of any type has absolutely no place in our society and will not be tolerated in USL matches," it added.
