Protein lysine 2-hydroxyisobutyrylation (Khib) is a newly identified post-translational modification found in animal and yeast cells. Previous research suggested that histone Khib is involved in male cell differentiation and plays a critical role in the regulation of chromatin functions in animals. However, information regarding protein Khib in plants is still limited. In this study, using a specific antibody and LC-MS/MS methods, we identified 11,976 Khib sites in 3,001 proteins in Physcomitrella patens. The bioinformatics analysis indicated that these Khib-modified proteins were involved in a wide range of molecular functions and cellular processes, and showed diverse subcellular localizations. Furthermore, an comparism of Khib sites in histone proteins among human, mouse and P. patens found conserved sites in the H3 and H4 histone proteins and novel sites in H1, H2A and H2B histone proteins in P. patens. This is the first report on Khib post-translational modifications in plants, and the study provides a comprehensive profile of Khib sites in histone and non-histone proteins in Physcomitrella patens.