Ketone bodies have long been known as a group of lipid-derived alternative energy sources during glucose shortages. Nevertheless, the molecular mechanisms underlying their non-metabolic functions remain largely elusive. This study identified acetoacetate as the precursor for lysine acetoacetylation (Kacac), a previously uncharacterized and evolutionarily conserved histone post-translational modification. This protein modification is comprehensively validated using chemical and biochemical approaches, including HPLC co-elution and MS/MS analysis using synthetic peptides, Western blot, and isotopic labeling. Histone Kacac can be dynamically regulated by acetoacetate concentration, possibly via acetoacetyl-CoA. Biochemical studies show that HBO1, traditionally known as an acetyltransferase, can also serve as an acetoacetyltransferase. In addition, 33 Kacac sites are identified on mammalian histones, depicting the landscape of histone Kacac marks across species and organs. In summary, this study thus discovers a physiologically relevant and enzymatically regulated histone mark that sheds light on the non-metabolic functions of ketone bodies.