Polarized growth plays a key role in all domains of their biology, including morphogenesis and pathogenicity of filamentous fungi. However, little information is available about the determinants of polarized growth. The fungal Mep2, Pes1, and Cph1 proteins were identified to be involved in the dimorphic transition between yeast and hyphal forms in Candida albicans. In this study, evidence that the dimorphic fungal entomopathogen Ophiocordyceps sinensis Mep2, Pes1, and Cph1 proteins are involved in polarized growth is presented. OsMep2 was significantly upregulated at aerial hyphae and conidia germination stages. OsCph1 was significantly upregulated at aerial hyphae, conidia initiation, and conidia germination stages, and OsPes1 was significantly upregulated at the conidia germination stage. Deletions of OsMep2, OsCph1, and OsPes1 provoked defects in the polarized growth. The abilities of hyphal formation and the yields of blastospores and conidia for the Δ OsMep2, ΔOsCph1, and Δ OsPes1 mutants were significantly reduced. The conidia yields of the ΔOsMep2, ΔOsCph1, and ΔOsPes1 mutants were decreased by 69.17%, 60.90%, and 75.82%, respectively. Moreover, the pathogenicity of the Δ OsMep2, ΔOsCph1, and Δ OsPes1 mutants against Thitarodes xiaojinensis was significantly reduced. The mummification rate caused by wide type and ΔOsMep2, ΔOsCph1, and ΔOsPes1 mutants were 36.98% ± 8.52%, 0.31% ± 0.63%, 1.15% ± 1.57%, and 19.69% ± 5.6%, respectively. These results indicated that OsMep2, OsCph1, and OsPes1 are involved in the regulation of hyphal formation, sporulation, and pathogenicity of O. sinensis. This study provided a basis for the understanding of the fungal dimorphic development and improving the efficiency of artificial cultivation of O. sinensis.