The Bayer Leverkusen midfielder was named in Joachim Low's provisional squad for the finals in Brazil after impressing during the Bundesliga season.
Germany are currently in northern Italy before heading to South America next month, but Bender will not be on the plane after the German Football Association confirmed he damaged a muscle in his right thigh during a training session.
"If just before a tournament injury fails a player then that's disappointing," said Low.
"For Lars, I'm personally very sorry.
"I know he wanted to be in Brazil, he is an absolute role model."
Bender has made 17 appearances for Germany since his debut in September 2011 and was part of Low's squad during the World Cup qualifying campaign.
The 25-year-old's withdrawal may see Low opt for Sami Khedira as part of his midfield options in Brazil, even though the Real Madrid player has been absent with a long-term knee injury this season.
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