The Manchester United star is sidelined with a shoulder injury and joins the likes of Michael Essien, Michael Ballack and John Mikel Obi in missing the tournament.
Pain game: Nani joins several big names on the sidelines
The Portugal federation said: 'After tests we conclude he is unfit to participate in theWorld Cup.'
Nani picked up the injury attempting an acrobatic shot in training on Friday in Lisbon. He will be replaced in the squad by Benfica midfielder Ruben Amorim.
The absence will disrupt the plans of Portugal boss Carlos Queiroz, who was counting on the 23-year-old following an impressive season at Old Trafford.
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The other main injury concern in the Portuguese squad is Pepe, who has been recovering from a knee injury he sustained in December.
He has not played since but was last week cleared by doctors from the national side and his club Real Madrid to travel to the finals.
Portugal play their first match on June 15 against the Ivory Coast in Group G, which also includes Brazil and North Korea.
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