Striker Radamel Falcao has failed to recover from knee surgery and was left out of Colombia's World Cup squad on Monday, said coach Jose Pekerman.
The 28-year-old ruptured the anterior cruciate ligament in his left knee while playing for Monaco in a French Cup match in January.
Falcao, nicknamed Tiger, was a key element of the Colombia squad that finished second to Argentina in the South American qualifying group, scoring nine goals in 13 matches.
The team's talisman was the player that Pekerman could least afford to lose as his side feature this month at their first finals for 16 years.
Experienced defender Luis Amaranto Perea, who has been recovering from injury, and forward Luis Muriel also missed the cut from Pekerman's provisional squad.
Colombia face Greece, Ivory Coast and Japan in Group C in Brazil later this month.