The ortholog of the human gene NPC1 was identified in the plant pathogenic, filamentous fungus Fusarium graminearum by shared amino acid sequence, protein domain structure and cellular localization of the mature fungal protein. The FusariumNpc1 gene shares 34% amino acid sequence identity and 51% similarity to the human gene, has similar domain structure and is constitutively expressed, although up-regulated in ungerminated macroconidia and ascospores. GFP-tagged Npc1p localizes to the fungal vacuolar membrane. Cultures derived from a ?npc1 mutant strain contain significantly more ergosterol than cultures of the wildtype. Staining with the fluorescent, sterol binding dye filipin, shows that ergosterol accumulates in vacuoles of the ?npc1 mutant but not the wildtype strain. The ?npc1 mutant has a temperature dependent reduction in growth and greater sensitivity to the ergosterol synthesis inhibiting fungicide tebuconazole compared with the wildtype strain or the mutant complemented with wildtype Npc1. The mutant also is significantly reduced in pathogenicity to wheat. Our results are consistent with the interpretation that Npc1p is important for normal transport of ergosterol from the vacuole and is essential for proper membrane function under particular environmental conditions.