Lysine benzoylation (Kbz) is a newly discovered protein post-translational modification (PTM). This PTM can be stimulated by benzoate and contributes to gene expression. However, its regulatory enzymes and substrate proteins remain largely unknown, hindering further functional studies. Here we identified and validated the lysine acetyltransferase (KAT) HBO1 as a “writer” of Kbz in mammalian cells. In addition, we report the first benzoylome in mammalian cells, identifying 1747 Kbz sites, among them at least 77 are the HBO1-targeted Kbz substrates. Bioinformatics analysis showed that HBO1-targeted Kbz sites were involved in multiple processes, including chromatin remodeling, transcription regulation, immune regulation, and tumor growth. Our results thus identify key regulatory elements of the Kbz pathway, and reveal new enzymatic activity and functions of HBO1 in cellular physiology.