Zambrotta played for the under-21 side for the 2000 Olympic Games in September. He started in the games against Australia and Honduras, which Italy won 1–0 and 3–1 respectively. Since they had already qualified for the quarter-finals, the coach put Zambrotta on the bench for the last game against Nigeria and substituted him on at the 80th minute. This would be the last time he played for the U21 national team, with a total of 13 U21-caps. Italy were defeated by Spain, who would go on to win the silver medal, by one goal in the quarter-final.