The in-form Manchester United winger suffered a shoulder injury after attempting an acrobatic shot in training on Friday in Lisbon.
But he still boarded the plane to South Africa and gave a thumbs-up on his arrival - sparking hopes he would be fine to play.
He told pals on the flight he would struggle to make Portugal's friendly with Mozambique yesterday, but was already feeling better.
But tests showed the injury was worse than first thought. Coach Carlos Queiroz moved quickly by calling up Benfica midfielder Ruben Amorim.
Nani, 23, feared he had suffered a dislocated shoulder during United's Community Shield clash with Chelsea last August but made a rapid recovery. But there was no such luck this time.
Portuguese FA chiefs said in a statement: "Following the injury a daily assessment of the damage was made and medical tests completed. We have concluded he is unable to play competitively."
Nani is expected to fly back to Manchester in the next few days and will join England's Rio Ferdinand in the United treatment room.
Portugal need all the help they can get in the so-called 'Group of Death' alongside Brazil, Ivory Coast and North Korea.
Nani impressed during the second half of the season for his club before scoring a spectacular lob in Portugal's friendly against Cameroon last week.
He is the latest English-based player to suffer World Cup heartache, following the likes of Chelsea's Michael Essien, Michael Ballack and John Obi Mikel.
His former United team-mate Cristiano Ronaldo started on the bench for the 3-0 friendly win over Mozambique at Wanderers cricket ground in Johannesburg.
Zenit St Petersburg's Danny stepped into Nani's shoes, scoring one of the goals in a man-of-the-match display.