Valencia was selected in Colombia's preliminary squad but he has been plagued by injury throughout the season, with the 29-year-old limited to just 25 minutes of football for Fluminense in Brazil's Serie A thus far.
And Colombia head coach Jose Pekerman confirmed Valencia, who has 11 international caps to his name, will not make the trip to Brazil, where Colombia are set to come up against Greece, Ivory Coast and Japan in Group C.
"I'll give some news that is not welcome and that requires us to say goodbye to Edwin Valencia who is dogged by injuries," Pekerman told reporters at Colombia's training camp in Argentina. "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 squad after Aquivaldo Mosquera (America), Elkin Soto (Mainz 05) and Macnelly Torres (Al Shabab) were overlooked last week.
However, there is some good news for Colombia with Pekerman prepared to give star striker Radamel Falcao up until June 2 - FIFA's deadline for the final 23-man squad - before making a decision on his participation at the World Cup.
Falcao is in a race against time to be fit for the World Cup after he suffered a serious knee injury in Monaco's Coupe de France tie in January, but Pekerman is giving the striker every chance to feature.
"We'll wait for Falcao until the last day, the last minute, until the last second that FIFA allows us," said the Argentine boss.
"We have a lot of people to make the final decision. We'll find the most logical reasons with the player and doctors."
