HBO1 catalyzes lysine lactylation and mediates histone H3K9la to regulate gene transcription. This study reveals that HBO1 functions as a lysine lactyltransferase, catalyzing the addition of lactyl groups to lysine residues on histones and other proteins. Quantitative proteomic analysis identified 95 endogenous Kla sites targeted by HBO1, with the majority located on histones. Site-specific antibodies confirmed that HBO1 preferentially catalyzes histone H3K9la, and scaffold proteins such as JADE1 and BRPF2 enhance its enzymatic activity. CUT&Tag assays showed that HBO1 is required for histone H3K9la at transcription start sites (TSSs), and regulated Kla promotes key signaling pathways and tumorigenesis. HBO1-KO cells exhibited reduced proliferation, migration, and invasion abilities, and lower levels of H3K9la in clinical tissues. These findings suggest that HBO1 regulates gene transcription through H3K9la-mediated mechanisms. The study also highlights the importance of understanding the biochemical processes involved in lactyl group transfer and removal, as well as the roles of writer and eraser proteins in lactylation. HBO1 is a member of the MYST family of histone acyltransferases and can catalyze various acylations, including acetylation, propionylation, butyrylation, crotonylation, and benzoylation. The study provides evidence that HBO1 is a lactyltransferase and plays a critical role in histone lactylation and gene regulation. The findings have implications for understanding the non-metabolic functions of lactate and its role in cancer development and progression.HBO1 catalyzes lysine lactylation and mediates histone H3K9la to regulate gene transcription. This study reveals that HBO1 functions as a lysine lactyltransferase, catalyzing the addition of lactyl groups to lysine residues on histones and other proteins. Quantitative proteomic analysis identified 95 endogenous Kla sites targeted by HBO1, with the majority located on histones. Site-specific antibodies confirmed that HBO1 preferentially catalyzes histone H3K9la, and scaffold proteins such as JADE1 and BRPF2 enhance its enzymatic activity. CUT&Tag assays showed that HBO1 is required for histone H3K9la at transcription start sites (TSSs), and regulated Kla promotes key signaling pathways and tumorigenesis. HBO1-KO cells exhibited reduced proliferation, migration, and invasion abilities, and lower levels of H3K9la in clinical tissues. These findings suggest that HBO1 regulates gene transcription through H3K9la-mediated mechanisms. The study also highlights the importance of understanding the biochemical processes involved in lactyl group transfer and removal, as well as the roles of writer and eraser proteins in lactylation. HBO1 is a member of the MYST family of histone acyltransferases and can catalyze various acylations, including acetylation, propionylation, butyrylation, crotonylation, and benzoylation. The study provides evidence that HBO1 is a lactyltransferase and plays a critical role in histone lactylation and gene regulation. The findings have implications for understanding the non-metabolic functions of lactate and its role in cancer development and progression.