Iniesta to miss Real Madrid opener
"Andreas Iniesta is out," Guardiola told reporters at a news conference at Real's Bernabeu stadium on Tuesday.
"He won't be there. One hundred percent," he added. "Maybe we will change formation."
Barca said in a statement on their website that the Spain international had a calf strain and had only been able to train for 10 minutes away from the rest of the team on Tuesday.
The creative Iniesta is typically deployed on the left side and is an important component of Barca's fast-flowing, attacking play.
Guardiola - already struggling with injuries to several defenders - could replace him with reserve player Thiago Alcantara, who has impressed on several recent appearances for the first team, or Netherlands international Ibrahim Afellay.