The midfielder has not played since April because of the issue, sustained in the Russian Premier League meeting between Kuban Krasnodar - who Shirokov joined on loan from Zenit in January - and Lokomotiv Moscow.
Fabio Capello included the 32-year-old in his 23-man squad despite the injury, but Shirokov has not been able to feature in any of Russia's World Cup warm-up matches.
And, following the 2-0 win over Morocco on Friday, coach Capello confirmed that Shirokov is due to have an operation in the next 48 hours and will miss the tournament as a result.
Shirokov's place in the squad will be taken by Rubin Kazan's Pavel Mogilevets, who had initially been named as Capello's standby player.
"On Monday, Shirokov will fly to Finland to undergo knee surgery there," Capello said.
"Shirokov is a very important member of Russia's squad but I hope that the rest of the players will be able to perform at their best level in Brazil anyway.
"Mogilevets will replace Shirokov in our squad but we still have time before June 15 to make final changes in the squad's line-up."
Russia begin their World Cup campaign against South Korea on June 18.
