Reus was injured in the first half of his country’s 6-1 friendly win over Armenia on Friday, and left the field in visible distress.
An initial statement from the German Football Association (DFB) on Saturday revealed the Borussia Dortmund forward had suffered a "partial tear of the anterior syndesmosis ligaments above the left ankle", but did not rule Reus out of the World Cup.
However, it has now been confirmed that the 25-year-old will miss the tournament, much to the disappointment of national coach Joachim Low.
"For him and us this is extremely regrettable," Low said in a statement on the DFB website.
"Marco was in great shape; he has left an excellent impression in training camp and in the two games against Cameroon and Armenia.
"In our considerations for Brazil, he played a central role."
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