Lysine 2-hydroxyisobutyrylation (K hib ) is a novel post-translational modification (PTM), which was thought to play a role in active gene transcription and cellular proliferation. Here we report a comprehensive identification of K hib in Proteus mirabilis (P. mirabilis). By combining affinity enrichment with two-dimensional liquid chromatography and high-resolution mass spectrometry, 4735 2-hydroxyisobutyrylation sites were identified on 1051 proteins in P. mirabilis. These proteins bearing modifications were further characterized in abundance, distribution and functions. The interaction networks and domain architectures of these proteins with high confidence were revealed using bioinformatic tools. Our data demonstrate that many 2-hydroxyisobutyrylated proteins are involved in metabolic pathways, such as purine metabolism, pentose phosphate pathway and glycolysis/gluconeogenesis. The extensive distribution of K hib also indicates that the modification may play important influence to bacterial metabolism. The speculation is further supported by the observation that carbon sources can influence the occurrence of K hib . Furthermore, we demonstrate that 2-hydroxyisobutyrylation on K343 was a negative regulatory modification on Enolase (ENO) activity, and molecular docking results indicate the regulatory mechanism that K hib may change the binding formation of ENO and its substrate 2-phospho- d -glycerate (2PG) and cause the substrate far from the active sites of enzyme. We hope this first comprehensive analysis of nonhistone K hib in prokaryotes is valuable for further functional investigation of this modification.