The study investigates the role of HBO1, a member of the MYST family of histone acetyltransferases, in lysine lactylation (Kla) and its impact on gene transcription. HBO1 is shown to function as a lysine lactyltransferase, catalyzing the addition of lactyl groups to lysine residues on histones both in vitro and in vivo. Key sites for lactyltransferase activity in HBO1 are identified, with E508 being crucial. Quantitative proteomics reveals 95 endogenous Kla sites, primarily on histones, with HBO1 preferentially catalyzing H3K9la. Scaffold proteins like JADE1 and BRPF2 enhance HBO1's lactyltransferase activity. CUT&Tag assays demonstrate that HBO1 is essential for H3K9la at transcription start sites (TSSs), and this modification promotes signaling pathways and tumorigenesis. HBO1 knockout cells exhibit reduced proliferation, migration, and invasion, and clinical cervical cancer tissues show higher levels of HBO1 and H3K9la compared to normal tissues. These findings highlight HBO1's role in mediating H3K9la-dependent gene regulation and its potential in tumorigenesis.The study investigates the role of HBO1, a member of the MYST family of histone acetyltransferases, in lysine lactylation (Kla) and its impact on gene transcription. HBO1 is shown to function as a lysine lactyltransferase, catalyzing the addition of lactyl groups to lysine residues on histones both in vitro and in vivo. Key sites for lactyltransferase activity in HBO1 are identified, with E508 being crucial. Quantitative proteomics reveals 95 endogenous Kla sites, primarily on histones, with HBO1 preferentially catalyzing H3K9la. Scaffold proteins like JADE1 and BRPF2 enhance HBO1's lactyltransferase activity. CUT&Tag assays demonstrate that HBO1 is essential for H3K9la at transcription start sites (TSSs), and this modification promotes signaling pathways and tumorigenesis. HBO1 knockout cells exhibit reduced proliferation, migration, and invasion, and clinical cervical cancer tissues show higher levels of HBO1 and H3K9la compared to normal tissues. These findings highlight HBO1's role in mediating H3K9la-dependent gene regulation and its potential in tumorigenesis.