Carotenoids are a structurally diverse class of terpenoid pigments that are synthesized by many microorganisms and plants. In this study, we identified five putative carotenoid biosynthetic genes from the ascomycete Gibberella zeae (GzCarB, GzCarO, GzCarRA, GzCarT, and GzCarX). HPLC showed that the fungus produces two carotenoids: neurosporaxanthin and torulene. We deleted the five genes individually to determine their functions. GzCarB, GzCarRA, and GzCarT were required for neurosporaxanthin biosynthesis, but the deletion of GzCarX or GzCarO (DeltagzcarX or DeltagzcarO) failed to alter the production of neurosporaxanthin or torulene. DeltagzcarRA and DeltagzcarB did not produce neurosporaxanthin or torulene. DeltagzcarB led to the accumulation of phytoene, which is an intermediate in carotenoid biosynthesis, but DeltagzcarRA did not. DeltagzcarT produced torulene but not neurosporaxanthin. Based on these functional studies and similarities to carotenoid biosynthesis genes in other fungi, we deduced the functions of the three genes and propose the carotenoid biosynthetic pathway of G. zeae.