In pathogenic fungi, N-glycosylated cell wall proteins serve as initial contact points during interactions with host. In the process of protein N-glycosylation, N-linked glycans are processed by α-glucosidases Ⅰ (Gls1) and Ⅱ (Gls2) to trim the final two glucose residues to facilitate glycoprotein maturation. In this study, we identified and characterized MaGls2 by disrupting its catalytic subunit MaGls2α, in the entomopathogenic fungus Metarhizium acridum. MaGls2α was found to localize to the endoplasmic reticulum. Deletion of MaGls2α resulted in earlier conidiation and increased conidial yield, but impaired colony growth and diminished tolerance to various stresses. ∆MaGls2α mutant exhibited disrupted cell wall architecture, characterized by a smoother surface, increased wall thickness, reduced β-1,3-glucan, and elevated chitin content. Bioassays demonstrated that the ∆MaGls2α mutant exhibited significantly attenuated virulence and triggered a stronger immune response from the host. Further analysis of the infection process revealed that reduced cell wall hydrophobicity and impaired appressorium formation in ∆MaGls2α mutant led to its decreased ability to penetrate the insect cuticle. Furthermore, the mutant showed slowed growth in host hemolymph, hindering successful colonization. These results indicate that MaGls2α is essential for maintaining proper cell wall composition and structure, thereby influencing fungal growth, conidiation, stress tolerance, and pathogenicity.