Lee, who won the continental award in 2012 after helping Ulsan Hyundai to the Asian Champions League title, suffered the injury playing for his club against Gyeongnam FC on Saturday and his withdrawal adds to a growing injury list for head coach Shin Tae-yong.
The 32-year-old's absence was confirmed by the Korea Football Association on Tuesday and he joins first-choice central defender Kim Min-jae of Jeonbuk Motors and Dijon winger Kwon Chang-hoon in pulling out of the squad due to injury.
Kim has been sidelined by a hairline fracture to his shin while Kwon will miss the World Cup after rupturing his Achilles.
There are also doubts over the fitness of Jeonbuk full back Kim Jin-soo, who has suffered a knee injury, and Shin will monitor the former Hoffenheim defender's form before deciding on his World Cup fate.
"Kim is doubtful to play our friendly matches next week," Shin said on Monday ahead of the start of the team's final World Cup preparations.
"If he doesn't meet the standards that we want, he is not going to be selected for the 23-player list."
(Reporting by Michael Church, Editing by Amlan Chakraborty)
