James took the top scorer award while representing the Cafeteros in 2014 when they reached the quarter-finals in Brazil, eventually going out to the hosts.
And four years later the No. 10 will again be at the forefront, as coach Jose Pekerman picked his preliminary list ahead of Russia.
Falcao, meanwhile, missed out on the last World Cup in heart-breaking fashion after rupturing his cruciate ligaments just days ahead of the tournament.
The likes of Yerry Mina, Davinson Sanchez and Juan Fernando Quintero, meanwhile, will hope to make the cut and debut at the finals in June.
Colombia preliminary World Cup squad:
Goalkeepers: David Ospina (Arsenal), Camilo Vargas (Deportivo Cali), Iván Arboleda (Banfield), José Fernando Cuadrado (Once Caldas)
Defenders: Cristian Zapata (Milan), Dávinson Sánchez (Tottenham), Santiago Arias (PSV Eindhoven), Óscar Murillo (Pachuca), Frank Fabra (Boca Juniors), Johan Mojica (Girona ), Yerry Mina (Barcelona), William Tesillo (Santa Fe), Bernardo Espinosa (Girona), Stefan Medina (Monterrey), Farid Díaz (Olimpia)
Midfielders: Wílmar Barrios (Boca Juniors), Carlos Sánchez (Espanyol), Jefferson Lerma (Levante), José Izquierdo (Brighton Hove & Albion), James Rodríguez (Bayern Múnich), Giovanni Moreno (Shanghái Shenhua), Abel Aguilar (Deportivo Cali), Mateus Uribe (América), Yimmi Chará (Júnior), Juan Fernando Quintero (River Plate), Edwin Cardona (Boca Juniors), Juan Guillermo Cuadrado (Juventus), Gustavo Cuéllar (Flamengo), Sebastián Pérez (Boca Juniors)