Summary The ergosterol biosynthesis pathway is well understood in S accharomyces cerevisiae , but currently little is known about the pathway in plant‐pathogenic fungi. In this study, we characterized the F usarium graminearum FgERG4 gene encoding sterol C ‐24 reductase, which catalyses the conversion of ergosta‐5,7,22,24‐tetraenol to ergosterol in the final step of ergosterol biosynthesis. The FgERG4 deletion mutant Δ FgErg4 ‐2 failed to synthesize ergosterol. The mutant exhibited a significant decrease in mycelial growth and conidiation, and produced abnormal conidia. In addition, the mutant showed increased sensitivity to metal cations and to various cell stresses. Surprisingly, mycelia of Δ FgErg4 ‐2 revealed increased resistance to cell wall‐degrading enzymes. Fungicide sensitivity tests revealed that Δ FgErg4 ‐2 showed increased resistance to various sterol biosynthesis inhibitors ( SBI s), which is consistent with the over‐expression of SBI target genes in the mutant. Δ FgErg4 ‐2 was impaired dramatically in virulence, although it was able to successfully colonize flowering wheat head and tomato, which is in agreement with the observation that the mutant produces a significantly lower level of trichothecene mycotoxins than does the wild‐type progenitor. All of these phenotypic defects of Δ FgErg4 ‐2 were complemented by the reintroduction of a full‐length FgERG4 gene. In addition, FgERG4 partially rescued the defect of ergosterol biosynthesis in the S accharomyces cerevisiae ERG4 deletion mutant. Taken together, the results of this study indicate that FgERG4 plays a crucial role in ergosterol biosynthesis, vegetative differentiation and virulence in the filamentous fungus F . graminearum .