Histone lysine lactylation is a physiologically and pathologically relevant epigenetic pathway that can be stimulated by the Warburg effect-associated L-lactate. Nevertheless, the mechanism by which cells use L-lactate to generate lactyl-coenzyme A (CoA) and how this process is regulated remains unknown. Here, we report the identification of guanosine triphosphate (GTP)-specific SCS (GTPSCS) as a lactyl-CoA synthetase in the nucleus. The mechanism was elucidated through the crystallographic structure of GTPSCS in complex with L-lactate, followed by mutagenesis experiments. GTPSCS translocates into the nucleus and interacts with p300 to elevate histone lactylation but not succinylation. This process depends on a nuclear localization signal in the GTPSCS G1 subunit and acetylation at G2 subunit residue K73, which mediates the interaction with p300. GTPSCS/p300 collaboration synergistically regulates histone H3K18la and GDF15 expression, promoting glioma proliferation and radioresistance. GTPSCS represents the inaugural enzyme to catalyze lactyl-CoA synthesis for epigenetic histone lactylation and regulate oncogenic gene expression in glioma.