Rosicky, who injured an Achilles tendon during Tuesday's 2-1 win against Greece and hasn't trained since, was ruled out hours before Saturday's game in Wroclaw, Poland. His place in the midfield went to Daniel Kolar.
"He was trying to run in the morning and it was still too painful for him to play," team spokesman Jaroslav Kolar said.
The loss of the Arsenal playmaker is a major blow to the Czechs' hopes of reaching the quarterfinals of a major tournament for the first time since 2004.
The winner of the match will qualify for the last eight. A draw for the Czech Republic would also be enough, provided Greece doesn't beat Russia by six goals or more in the group's other game.
Rosicky injured a calf muscle in Arsenal's last Premier League match on May 13 and only recovered last week.
He also missed Euro 2008 due to injury.