Chronic systemic inflammation is a pathological manifestation of metabolic disorders, leading to elevated glycolytic flux and a shift towards aerobic glycolysis and lactate generation. This increase in lactate corresponds with increased generation of lactoyl/Lys modifications on histones, which mediate transcriptional responses to inflammatory stimuli. Lactoylation is also generated through a non-enzymatic S-to-N acyltransfer from the glyoxalase cycle intermediate, lactoylglutathione (LGS). This study reports that LGS plays a regulatory role in mediating histone lactoylation and inflammatory signaling. In the absence of the primary LGS hydrolase, glyoxalase 2 (GL2), RAW264.7 macrophages display significant elevations in LGS and histone lactoylation, leading to a potentiated inflammatory response when exposed to lipopolysaccharides. Chromatin accessibility analysis shows that lactoylation is associated with more compacted chromatin than acetylation in an unstimulated state, but regions of the genome associated with lactoylation become more accessible upon stimulation. Additionally, the study demonstrates a spontaneous S-to-S acyltransfer of lactate from LGS to CoA, yielding lactoyl-CoA. This represents the first known mechanism for the generation of this metabolite. Collectively, these data suggest that LGS, rather than intracellular lactate, is the primary driving factor facilitating histone lactoylation and a major contributor to inflammatory signaling.Chronic systemic inflammation is a pathological manifestation of metabolic disorders, leading to elevated glycolytic flux and a shift towards aerobic glycolysis and lactate generation. This increase in lactate corresponds with increased generation of lactoyl/Lys modifications on histones, which mediate transcriptional responses to inflammatory stimuli. Lactoylation is also generated through a non-enzymatic S-to-N acyltransfer from the glyoxalase cycle intermediate, lactoylglutathione (LGS). This study reports that LGS plays a regulatory role in mediating histone lactoylation and inflammatory signaling. In the absence of the primary LGS hydrolase, glyoxalase 2 (GL2), RAW264.7 macrophages display significant elevations in LGS and histone lactoylation, leading to a potentiated inflammatory response when exposed to lipopolysaccharides. Chromatin accessibility analysis shows that lactoylation is associated with more compacted chromatin than acetylation in an unstimulated state, but regions of the genome associated with lactoylation become more accessible upon stimulation. Additionally, the study demonstrates a spontaneous S-to-S acyltransfer of lactate from LGS to CoA, yielding lactoyl-CoA. This represents the first known mechanism for the generation of this metabolite. Collectively, these data suggest that LGS, rather than intracellular lactate, is the primary driving factor facilitating histone lactoylation and a major contributor to inflammatory signaling.