Protein phosphatase 2A is a subgroup of widely conserved serine/threonine phosphatases and plays diverse roles in transcription, translation, differentiation, cell cycle, and signal transduction in many organisms. However, its roles in biotrophic and hemi-biotrophic phytopathogenic fungi remain to be investigated. In this study, we isolated an insertional mutant of the rice blast fungus Magnaporthe oryzae that was defective in vegetative hyphal growth. In the mutant, the T-DNA fragment was found to be inserted in the promoter region of a putative serine/threonine protein phosphatase 2A catalytic subunit (PP2Ac) gene MoPPG1. Deletion of MoPPG1 leads to severe defects in vegetative hyphal growth and conidiation. Conidia of the ∆Moppg1 null mutants were misshaped, and most of them were two-celled. The deletion mutants of MoPPG1 did not penetrate into host plant cells and failed to cause any disease lesions on rice leaves. Interestingly, significant reduction was found in the ∆Moppg1 null mutants in expression levels of several Rho GTPase family genes including MgCDC42, MgRHO3, and MgRAC1, which were important for pathogenesis of M. oryzae. Taken together, our results indicated that PP2Ac plays vital roles in asexual development and plant infection by regulating Rho GTPases in the rice blast fungus and perhaps other plant pathogenic fungi.