Ketogenesis derived BHB facilitates 3-hydroxybutyrylation on lysine linking metabolism with epigenetic regulation. Herein, we found HBO1 served as 3-hydroxybutyryltransferase preferentially catalyzing lysine 9 of histone H3 (H3K9bhb). Moreover, total 1171 Kbhb sites on 582 proteins were identified. Collectively, we firstly descibed HBO1 mediated Kbhb regulatory network in HeLa cells.