By the combination of affinity enrichment and high-resolution LC-MS/MS analysis, large-scale lysine 2-hydroxyisobutyrlome analysis was performed in Oryza Sativa. Altogether, 9916 lysine 2-hydroxyisobutyrylation sites in 2512 protein groups were identified. Intensive bioinformatic analysis was then carried out to annotate those lysine 2-hydroxyisobutyrylated targets, including protein annotation, functional classification, functional enrichment, functional enrichment-based cluster analysis, etc. Based on the results, further studies following the large-scale lysine 2-hydroxyisobutyrylome analysis were suggested.