Timlin made his first appearance with the Cardinals (and in the National League) on July 30, 2000, pitching one inning in relief against the New York Mets. He made 25 total appearances with the Cardinals during the regular season; he had a 3–1 record, with one save and a 3.34 ERA. He appeared in two games of the National League Division Series (NLDS) against the Atlanta Braves, and in three games of the National League Championship Series (NLCS) against the Mets. He took the loss in Game 2 of the NLCS, giving up an unearned run while pitching the ninth inning.