Iran's team sang the national anthem before Friday's World Cup match against Wales, having opted not to do so in their tournament opener in Qatar.
The Iranian players had stood impassively during their anthem before their 6-2 defeat to England on Monday in an apparent gesture of solidarity with anti-government protesters in the Islamic republic.
An AFP photographer at the stadium on Friday witnessed security staff confiscating a flag from a fan with the protest slogan "Woman, Life, Freedom".
Some Iranian fans were in tears during the singing of the anthem.
Iran forward Mehdi Taremi denied on Thursday that his team had come "under pressure" from their government to sing the anthem at the World Cup.
"I don't like to talk about political issues, but we are not under any pressure," Taremi said on the eve of the Wales game.
Iran has been shaken by two months of nationwide protests since the death of 22-year-old Mahsa Amini in morality police custody on 16 September.
Amini, a 22-year-old Iranian of Kurdish origin, died three days after her arrest in Tehran over an alleged breach of the dress code for women, which includes the mandatory hijab headscarf.
The crackdown since Amini's death has left at least 400 people dead, according to Oslo-based group Iran Human Rights.
The state's response has led to questions over whether the team represents Iran or the regime that has ruled with an iron fist since the Islamic Revolution of 1979.