Fluminense midfielder Edwin Valencia has been trimmed from Colombia's preliminary squad after failing to recover from injury.
National coach Jose Pekerman confirmed on Wednesday that the 29-year-old will not be fit in time for this summer's showpiece event in Brazill, having struggled with since since the start of the season.
The Argentina-born boss told reporters at the squad's training camp in Buenos Aires: "Edwin Valencia will not go to the World Cup.
"The injuries and problems Valencia has carried do not allow him to reach the form the national team needs in the World Cup.
"We are forced to leave him out, considering that he will not make it in the best physical condition."
Pekerman added: "This is not good news at all.
"Physical fitness is a very important factor and we have to know which players are fully fit to play the entire World Cup."
Valencia is the fourth player to be cut from the provisional group after America defender Aquivaldo Mosquera, Mainz midfielder Elkin Soto and Al Shabab midfielder Macnelly Torres missed out last week.
Despite having omitted Valencia due to fitness worries, Pekerman vowed to give Monaco striker Radamel Falcao until the last moment to prove he was back to full health having torn cruciate ligaments in his left knee in January.
"We'll wait for Falcao until the last day, the last minute," the Colombia coach said.
"We have a lot of people to make the final decision. We'll find the most logical reasons with the player and doctors."
Colombia squad: David Ospina (Niza), Farid Mondragon (Deportivo Cali), Camilo Vargas (Santa Fe); Camilo Zuniga (Napoli), Pablo Armero (West Ham), Cristian Zapata, Mario Yepes (Atalanta), Luis Amaranto Perea (Cruz Azul), Carlos Valdes (San Lorenzo), Santiago Arias, Eder Alvarez-Balanta (River Plate); James Rodriguez (Monaco), Abel Aguilar (Toulouse), Carlos Sanchez (Elche), Fredy Guarin (Inter Milan), Juan-Fernando Quintero (Porto), Aldo Ramirez (Morelia), Alex Mejia (Atletico Nacional), Victor Ibarbo (Cagliari), Juan-Guillermo Cuadrado (Fiorentina); Jackson Martinez (Porto), Radamel Falcao (Monaco), Teofilo Gutierrez (River Plate), Luis Fernando Muriel (Udinese), Carlos Bacca (Sevilla), Adrian Ramos (Borussia Dortmund).