β-Hydroxybutyrylation (kbhb) is a new type of post-translational modification(PTM) of lysine acylation. In recent years, kbhb modification has been shown to be involved in energy metabolism, tumor metabolism, DNA damage repair and other processes. Currently, lysine β-hydroxybutyrylation has not been studied in cardiac tissue. In this study, we performed proteomic and β-hydroxybutyrylation modification omics analysis of mouse heart tissue based on LC-MS/MS analysis. These data provide the first mammalian cardiac kbhb dataset and provide new directions for further investigation of the function of kbhb in non-histone proteins.