erg4is a key gene for ergosterol biosynthesis in filamentous fungi, but its function inPenicillium expansumremains unknown. Our results showed thatP. expansumcontains threeerg4genes, includingerg4A,erg4Banderg4C. The expression levels of the three genes showed differences in the wild-type (WT) strain, and the expression level oferg4Bwas the highest, followed byerg4C. Deletion oferg4A,erg4Borerg4Cin the WT strain revealed functional redundancy between them. Compared to the WT strain,erg4A,erg4Borerg4Cknockout mutants reduced ergosterol levels, witherg4Bdeletion having the greatest effect. Furthermore, deletion of the three genes reduced sporulation of the strain, and Δerg4Band Δerg4Cmutants showed defective spore morphology. In addition, Δerg4Band Δerg4Cmutants were found to be more sensitive to cell wall integrity and oxidative stress. However, deletion oferg4A,erg4Borerg4Chad no significant effect on colony diameter, spore germination rate, conidiophore structure ofP. expansumor pathogenicity to apple fruit. Taken together,erg4A,erg4Banderg4Chave redundant functions and are all involved in ergosterol synthesis and sporulation inP. expansum. In addition,erg4Banderg4Ccontribute to spore morphogenesis, cell wall integrity and response to oxidative stress inP. expansum.