Colombia beat Venezuela 1-0 on Wednesday (AEDT), with James scoring the winner from the penalty spot.
However, Peru beat Paraguay to finish one point above Colombia in the CONMEBOL standings, meaning it took fifth place and will go up against either Australia or the United Arab Emirates in an inter-confederation play-off.
James, who shot to stardom with his standout performances for Colombia at the 2014 World Cup when winning the Golden Boot, plays his club football in Qatar, yet one of South American football's most prominent poster boys will not be at the showpiece tournament.
It is a fall from grace for Colombia, who reached the quarter-finals in 2014 and the last 16 in 2018, having failed to qualify for the 2002, 2006 and 2010 editions of the tournament, while they also finished third in the 2021 Copa America.
Former Real Madrid and Bayern Munich playmaker James will be 34 by the time the 2026 World Cup comes around, meaning he may have missed his last chance to represent his country on the biggest stage.
"The most beautiful thing that has happened to me in my career as a footballer was attending two World Cups representing the shirt that I love the most, the yellow of my national team," he wrote on social media.
Lo más lindo que me ha pasado en mi carrera como futbolista, fue el asistir a dos mundiales representando la camiseta que más amo, la amarilla de mi selección. Hoy tengo un dolor profundo. Considero que nuestro país tiene talento y jugadores para siempre hacer parte del Mundial. pic.twitter.com/r3Sp59w6wz— James Rodríguez (@jamesdrodriguez) March 30, 2022
"Today I am in deep pain. I believe that our country has the talent and players to always be part of the World Cup.
"I feel sad and not only for myself, but also for my colleagues who deserve to shine and I know they have how. I don't know if [I'm] coming for the next processes, I don't know if [I'll be] here or not.
"What I do know is that it breaks my soul to lose, it makes me uncomfortable not being classified and this cannot happen again."
James, who disputed coach Reinaldo Rueda's reasoning as to why he was not selected for last year's Copa America, also suggested Colombia must reassess after their failure.
"I want Colombia to go to all the World Cups," he wrote.
"We must plan, join forces and work hard from the administrative and sports aspects to once again show the world the power and talent of our land."