Lysine 2-hydroxyisobutyrylation (Khib) 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 Khib in the rice false smut fungus Ustilaginoidea vi-rens, using Tandem Mass Tag (TMT)-based quantitative proteomics approach. A total of 3426 Khib sites were identified in 977 proteins, sugg esting that Khib 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 inter-connected 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 en-zyme that removes 2-hydroxyisobutyrylation and acetylation in U. virens. Notably, mutations of Khib sites in the mitogen-activated protein kinase (MAPK) UvSlt2 significantly reduced fungal virulence and decreased the enzymatic activity of UvSlt2. Molec-ular dynamics simulations demonstrated that 2-hydroxyisobutyrylation in UvSlt2 increased the hydrophobic solvent-accessible surface area and thereby affected binding between the UvSlt2 en-zyme and its substrates. Our findings thus establish Khib as a major post-translational modification in U. virens and point to an important role for Khib in the virulence of this phytopathogenic fungus.