Side judge Brian Stropolo has been replaced by an alternate, Tim Keese. The NFL made the decision Sunday morning just hours before kickoff.
"He was replaced because of the information that surfaced disclosing that he is a Saints fan," league spokesman Michael Signora said.
The league will look further into the matter. It received several phone calls and emails about Stropolo, who is from New Orleans and posted several photos of himself in Saints gear tailgating at a preseason game on his Facebook page.
Stropolo worked the Week 1 NFL opener between the Cowboys and Giants.
ESPN was first to report the news.
The NFL locked out the regular officials in June after their contract expired. Negotiations with the NFL Referees Association broke down several times during the summer, including just before the season, and the league is using replacements for the first time since 2001.
Back then, the lockout lasted for one week of the regular season before a settlement was reached. This is the second weekend the replacements are being used, and the NFL has drawn up a five-week schedule for using them if the labor dispute is not resolved.
Last week, there was one major error by the replacement officials, when they awarded Seattle an extra timeout in the final minutes of a game at Arizona. The Cardinals held on to win and the crew's referee admitted the mistake after the game.