Abstract: The post-translational modification lysine 2-hydroxyisobutyrylation (K_hib) plays an important role in gene transcription, metabolism, and enzymatic activity. K_hib sites have been identified in rice (Oryza sativa). However, the K_hib status of proteins in rice flowers during pathogen infection remains unclear. Here, we report a comprehensive identification of K_hib-modified proteins in rice flowers, and the changes in these proteins during infection with the fungal pathogen Ustilaginoidea virens. By using a tandem mass tag-based quantitative proteomics approach, we identified 2,891 K_hib sites on 964 proteins in rice flowers. Our data demonstrated that 2-hydroxyisobutyrylated proteins are involved in diverse biological processes. K_hib levels were substantially reduced upon infection with U. virens. Chromatin immunoprecipitation polymerase chain reaction (PCR) and reverse transcription quantitative PCR analyses revealed that histone K_hib is involved in the expression of disease-resistance genes. More importantly, most quantified sites on core histones H3 were downregulated upon U. virens infection. In addition, the histone deacetylases HDA705, HDA716, SRT1, and SRT2 are involved in the removal of K_hib marks in rice. HDA705 was further confirmed to negatively regulate rice disease resistance to pathogens U. virens, Magnaporthe oryzae, and Xanthomonas oryzae pv. oryzae (Xoo). Our data suggest that U. virens could modulate K_hib in rice flowers during infection.