Opy2 is an important membrane-anchored protein upstream of the HOG-MAPK signaling pathway and plays important roles in both the HOG-MAPK and Fus3/Kss1 MAPK. In this study, the roles of MaOpy2 in Metarhizium acridum were systematically elucidated. The results showed that the MaOpy2 disruption significantly reduced fungal tolerances to UV, heat shock and cell-wall-disrupting agents. Bioassays showed that the decreased fungal pathogenicity by topical inoculation mainly resulted from the impaired penetration ability. However, the growth ability of ∆MaOpy2 was enhanced in insect hemolymph. Importantly, MaOpy2 deletion could significantly increase the conidial yield of M. acridum by shifting the conidiation pattern from normal conidiation to microcycle conidiation on the 1/4SDAY medium. Sixty-two differentially expressed genes (DEGs) during the conidiation pattern shift, including 37 up-regulated genes and 25 down-regulated genes in ∆MaOpy2, were identified by RNA-seq. Further analysis revealed that some DEGs were related to conidiation and hyphal development. This study will provide not only the theoretical basis for elucidating the regulation mechanism for improving the conidial yield and quality in M. acridum but also theoretical guidance for the molecular improvement of entomopathogenic fungi.