Update publication information. Lysineβ-hydroxybutyrylation (kbhb), a novel protein post-translational modification (PTM) of lysine residues withβ-hydroxybuty group, is associated with ketone metabolism in numerous species. However, its potential role in diabetes, especially in diabetic cardiomyopathy (DCM), remains largely unexplored. In this study, we performed antibody-based affinity enrichment and liquid chromatography-mass spectrometry (LC-MS/MS) to analyze the kbhb residues on heart tissues of both wild types and established DCM model rat quantitatively. A total of 3520 β-hydroxybutyrated lysine sites in 1089 proteins were identified in this study. Further analysis showed that 336 kbhb sites in 143 proteins were differentially expressed between the heart tissues of DCM and wild-type rats. Among them, 284 Kbhb sites corresponding to 96 proteins increased, while 52 corresponding to 47 proteins decreased. Bioinformatic analysis of the proteomic results showed the up-regulated proteins in the heart tissues of DCM rats to be primarily located in the cytoplasm. In contrast, the down-regulated kbhb proteins were distributed mainly in the mitochondria. Functional enrichment analysis showed that the up-regulated proteins were enriched in the tricarboxylic acid cycle, oxidative phosphorylation, and propanoate metabolism. In contrast, the down-regulated proteins were enriched in the cGMP-PKG signaling pathway and glutathione metabolism. Our findings demonstrated how kbhb is related to many metabolic pathways, especially in those involved in energy metabolism. This study provides the first global investigation of the kbhb profile in DCM progression and can be an essential resource to explore DCM's pathogenesis further.