"After bruising his collarbone and after medical tests, we have concluded the player is unfit to play the World Cup," an FPF statement read.
"The medical dossier will be presented to Manchester United's medical department," the FPF added.
Nani, four goals in 34 games for his country to date - including three during qualifying - suffered the injury in training on Friday and the 23-year-old will now be replaced by Ruben Amorim of Benfica.
Under FIFA rules, a player can be replaced until the day before their first match.
Portugal, who go up against Brazil, Ivory Coast and North Korea, were playing a friendly later in Johannesburg against Mozambique.
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