The pair were driving to the city of Barquisimeto when they attempted to avoid an object in the road and crashed. Bandits then robbed the pair, officials said.
Valbuena, 33, and Castillo, 37 were teammates for Cardenales de Lara, a team playing in the Venezuelan Winter League, and were heading home after a game.
The news was relayed by Yaracuy state Governor Julio Leon Heredia.
Local officials explained that the circumstances surrounding the incident were consistent with tactics employed by bandits in the region.
They place or throw objects into the road to force cars to veer off the road or crash, then they approach the stricken vehicle and rob the occupants.
Heredia confirmed that four suspects have been detained after being discovered in possession of the players’ stolen property.
According to local reports, another player, third baseman Carlos Rivero, was also in the car, but survived the incident.
Valbuena was an 11-year Major League Baseball veteran who enjoyed stints at the Los Angeles Angels, Seattle Mariners, Cleveland Indians, Chicago Cubs and Houston Astros.
His former manager at the Astros, AJ Hinch, issued a statement saying: “I will miss Luis’ banter, smile, genuine love for his teammates, and, of course, the bat flips.
“He was a beloved person whether he was on our team or across the field.”
Those sentiments were echoed by Astros general manager Jeff Luhnow, whose statement said: “Luis was always smiling and was one of the happiest players in baseball.
“He provided joy to his teammates and our fans. He helped our franchise turn a corner in 2015 and provided many cherished memories.”
Castillo was a five-year veteran, representing the Pittsburgh Pirates, San Francisco Giants and the Houston Astros.
Major figures have spoken about the tragic incident, with Venezuelan president Nicolas Maduro tweeting: “I join in the mourning that has overwhelmed the Venezuelan baseball family and all of its fans.”
Commissioner of Major League Baseball Rob Manfred also issued a statement, saying it was “a very sad day for our sport.”
“It is clear by all the stories today that they loved baseball and made an impact on their teammates and the clubs they represented,” he said, expressing “my deepest condolences to their families, friends and fans, particularly those in their native Venezuela.”
The Venezuelan Professional Baseball League announced that their scheduled games on Friday were cancelled, while all the league’s stadiums will fly their flags at half-mast for three days in remembrance.
Venezuelan teams often travel together on team buses flanked by security forces to ensure the players’ safety.
As a result of the tragic incident involving Castillo and Valbuena the league are now reviewing their policy of allowing players to travel independently of their teams, with league president Juan Jose Avila suggesting in a radio interview that if the pair had travelled on the team bus “nothing would have happened to them.”