The oryzapsin genes opsA and opsB in Aspergillus oryzae encoding glycosylphosphatidylinositol (GPI)-anchored aspartic endopeptidase are homologs of Saccharomyces cerevisiae yapsins. We recently found another homolog, opsC, in the A. oryzae genome database, which was suggested to be a pseudogene. However, the profiles and roles of the proteins encoded by these genes have not yet been clarified. Toward this end, we first produced opsA- and opsB-overexpression strains and performed enzymatic analyses, revealing that OpsA and OpsB can attack sites other than the carboxyl-terminal peptide bonds of basic amino acids. Moreover, OpsA and OpsB were confirmed to bind to the cell membrane with a GPI anchor. Second, opsA and opsB single-deletion and double-deletion strains (Delta opsA, Delta opsB, and Delta opsA Delta opsB) were constructed to explore the expected roles of oryzapsins in cell wall synthesis, similar to the role of yapsins. The transcription level of mpkA in the cell wall integrity pathway was increased in Delta opsB and Delta opsA Delta opsB strains, suggesting that OpsB might be involved in processing cell wall synthesis-related proteins. Treatment with an ergosterol biosynthesis inhibitor reduced the growth of the Delta opsA Delta opsB strain. Moreover, the mRNA levels of Aoerg1, Aoerg3-1, Aoerg3-2, Aoerg7b, Aoerg11, and Aohmg1,2 showed a decreasing tendency in the Delta opsA Delta opsB strain, and the ergosterol content in the membrane was reduced in the Delta opsA Delta opsB strain. These results suggest that oryzapsins exist in the cell membrane and play roles in the formation of cell membranes. This is the first report of the involvement of GPI-anchored aspartic endopeptidases in ergosterol biosynthesis.