Colombia will be without first-choice left-back Frank Fabra at the World Cup after he suffered an anterior cruciate ligament rupture.
The Boca Juniors defender underwent examinations on Saturday after sustaining an injury to his left knee at Colombia's training camp in Italy.
ACL damage was confirmed by the Colombian Football Federation (FCF), who are yet to name a replacement for Fabra in their 23-man squad.
"The Colombian team reports with regret that Frank Fabra suffered a rupture of the anterior cruciate ligament of the left knee," an FCF statement read.
"After diagnostic tests of the defender [on Saturday], the medical team confirmed the injury that was expected.
"The entire squad, coaching team and staff received confirmation of the news with great pain."
Colombia coach Jose Pekerman had previously taken a gamble on the fitness of two players when he named his initial 23-man squad for the tournament.
Deportivo Cali midfielder Abel Aguilar, 33, and AC Milan defender Cristian Zapata, 31, both received the nod from Pekerman despite facing fitness problems.
They missed the 0-0 friendly draw with Egypt last Friday, but the Colombian Football Federation confirmed the experienced duo had "intensified" their rehabilitation in an attempt to be ready for Russia.
Zapata's availability means there was no place for Santa Fe youngster William Tesillo, while long-serving forward Teofilo Gutierrez also missed out.
Colombia drew 0-0 with Egypt in their only warm-up friendly last week and begin their World Cup campaign against Japan on June 19, with Group H matches against Poland and Senegal to follow.
Colombia's final 23-man World Cup squad in full, prior to the injury to Fabra:
Goalkeepers: Jose Cuadrado (Once Caldas), David Ospina (Arsenal), Camilo Vargas (Deportivo Cali);
Defenders: Santiago Arias (PSV), Frank Fabra (Boca Juniors), Yerry Mina (Barcelona), Johan Mojica (Girona), Oscar Murillo (Pachuca), Davinson Sanchez (Tottenham), Cristian Zapata (AC Milan);
Midfielders: Abel Aguilar (Deportivo Cali), Wilmar Barrios (Boca Juniors), Juan Cuadrado (Juventus), Jefferson Lerma (Levante), Juan Quintero (River Plate), James Rodriguez (Bayern Munich), Carlos Sanchez (Espanyol), Mateus Uribe (Club America);
Forwards: Miguel Borja (Palmeiras), Radamel Falcao (Monaco), Jose Izquierdo (Brighton and Hove Albion), Luis Muriel (Sevilla).