Hydrogen sulfide (H2S), a potent signaling molecule, regulates cellular activities in plants and mammals through S-sulfhydration, a post-translational modification. The role of H2S and its molecular targets in fungi, however, remain unclear. Our study demonstrates that H2S, synthesized by cystathionine γ-lyase (CSE1) in the rice blast fungus Magnaporthe oryzae, is essential for optimal fungal infection. Excessive H2S, through S-sulfhydration, impairs fungal infectivity by negatively regulating autophagy. Using quantitative proteomics, we identified numerous S-sulfhydrated proteins in M. oryzae, including the autophagy-related protein ATG18. We found that S-sulfhydration of a critical cysteine residue (Cys78) in ATG18 is essential for its binding to phosphatidylinositol 3-phosphate (PtdIns(3)P), thereby maintaining the protein's structural stability and regulating autophagy. This study uncovers a new mechanism by which H2S-mediated S-sulfhydration controls autophagy in pathogenic fungi, and suggests the potential use of H2S donors as a strategy to control fungal diseases by targeting fungal development and infection structures, with broad implications for agriculture and disease management.