Abstract: Lysine 2‐hydroxyisobutyrylation (K_hib) is a newly identified post‐translational modification (PTM) that plays important roles in transcription and cell proliferation in eukaryotes. However, its function remains unknown in phytopathogenic fungi. Here, we performed a comprehensive assessment of K_hib in the rice false smut fungus Ustilaginoidea virens, using Tandem Mass Tag (TMT)‐based quantitative proteomics approach. A total of 3 426 K_hib sites were identified in 977 proteins, suggesting that K_hib is a common and complex PTM in U. virens. Our data demonstrated that the 2‐hydroxyisobutyrylated proteins are involved in diverse biological processes. Network analysis of the modified proteins revealed a highly interconnected protein network that included many well‐studied virulence factors. We confirmed that the Zn‐binding reduced potassium dependency3‐type histone deacetylase (UvRpd3) is a major enzyme that removes 2‐hydroxyisobutyrylation and acetylation in U. virens. Notably, mutations of K_hib sites in the mitogen‐activated protein kinase (MAPK) UvSlt2 significantly reduced fungal virulence and decreased the enzymatic activity of UvSlt2. Molecular dynamics simulations demonstrated that 2‐hydroxyisobutyrylation in UvSlt2 increased the hydrophobic solvent‐accessible surface area and thereby affected binding between the UvSlt2 enzyme and its substrates. Our findings thus establish K_hib as a major post‐translational modification in U. virens and point to an important role for K_hib in the virulence of this phytopathogenic fungus.