Iran captain Masoud Shojaei and midfielder Ehsan Haji Safi actively played for their Europa League team, Greece's Panionios, when it faced Maccabi Tel Aviv last week.
Shojaei and Haji Safi started and played the full 90 minutes for Panionios, a club in the Greek Super League, in an August 3 match against Maccabi Tel Aviv.
As a result, and following a a promise to "thoroughly examine the full extent of the matter", Deputy Sports Minister Mohammad Reza Davarzani said they will never wear the national shirt again, telling state television: "Shojaei and Haji Safi have no place in Iran's national football team any more because they crossed the country's red line".
Photo Iran's Ehsan Haji Safi, right, during a match against Nigeria.
Shojaei - who has earned 70 worldwide caps - was instrumental in helping Iran secure a spot at the 2018 World Cup.
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According to ESPN FC, Iran prohibits its athletes from facing Israeli opponents since the Iranian government doesn't recognize Israel as a sovereign nation. "They can be replaced, but that is not an ideal situation".
"I am wholeheartedly hoping for Federation Internationale de Football Association to deprive Iran from participating in the World Cup for banning Shojaei and Haji Safi", said another Iranian soccer fan, Hushang Mirza, on Twitter, referring to soccer's world governing body.
Shojaei was also ostracized in June when he asked Irani president Hassan Rouhani if women could enter the stadium to watch the team play.
Their absences will come at a bad time for Iran.