Zambrano, who has maintained since January that he would not participate in the event, was included on the Venezuela roster released Tuesday.
"No, I'm staying here," Zambrano said Wednesday.
The Cubs right-hander has been having problems with his right eye, and he was to be examined this week. Zambrano may undergo Lasik surgery this spring. He is tentatively scheduled to start Monday when the Cubs play host to Arizona at HoHoKam Park.
A three-time All-Star, Zambrano did pitch in two games in the 2006 World Baseball Classic, going 1-0 with a 5.40 ERA. Zambrano is coming off a 14-6 season, which included his first no-hitter.
The Cubs do have two regulars participating in the event. Ted Lilly will pitch for Team USA and is expected to start the third game, while catcher Geovany Soto, who won the National League Rookie of the Year Award in 2008, will play for Puerto Rico. Soto expects to be the designated hitter in the first game and catch in the third.
Cubs pitcher Carlos Marmol, who was on the Dominican Republic's provisional roster, also has decided not to participate in the World Baseball Classic. He did hear Tuesday from Dominican manager Felipe Alou, who tried to get the Cubs pitcher to change his mind.
Carrie Muskat is a reporter for MLB.com. This story was not subject to the approval of Major League Baseball or its clubs.