Lactoylglutathione (LGSH) promotes inflammatory signaling in macrophages through histone lactoylation. This study reveals that LGSH, a glyoxalase cycle intermediate, serves as a key regulator of histone lactoylation and inflammatory responses. In GLO2 knockout (KO) RAW264.7 macrophages, LGSH levels increase significantly, leading to enhanced inflammatory responses to lipopolysaccharide (LPS) mimetic Kdo₂-Lipid A. This is accompanied by increased histone lactoylation, which is associated with chromatin compaction in unstimulated cells but becomes more accessible upon stimulation. The study also demonstrates that LGSH can undergo a non-enzymatic S-to-S acyltransferase reaction to generate lactoyl-CoA, a novel mechanism for lactoyl-CoA production. These findings suggest that LGSH, rather than intracellular lactate, is the primary driver of histone lactoylation and a major contributor to inflammatory signaling in macrophages. The data indicate that histone lactoylation is site-specific and regulated by GLO2, with significant increases in specific histone marks like H3K18lac in KO cells. Chromatin accessibility analysis using ATAC-seq shows that GLO2 knockout leads to widespread changes in chromatin accessibility, with increased accessibility at specific genomic loci. These changes are associated with increased H3K18lac, suggesting that histone lactoylation plays a role in gene activation. Overall, the study highlights the importance of LGSH in regulating inflammatory signaling through histone lactoylation.Lactoylglutathione (LGSH) promotes inflammatory signaling in macrophages through histone lactoylation. This study reveals that LGSH, a glyoxalase cycle intermediate, serves as a key regulator of histone lactoylation and inflammatory responses. In GLO2 knockout (KO) RAW264.7 macrophages, LGSH levels increase significantly, leading to enhanced inflammatory responses to lipopolysaccharide (LPS) mimetic Kdo₂-Lipid A. This is accompanied by increased histone lactoylation, which is associated with chromatin compaction in unstimulated cells but becomes more accessible upon stimulation. The study also demonstrates that LGSH can undergo a non-enzymatic S-to-S acyltransferase reaction to generate lactoyl-CoA, a novel mechanism for lactoyl-CoA production. These findings suggest that LGSH, rather than intracellular lactate, is the primary driver of histone lactoylation and a major contributor to inflammatory signaling in macrophages. The data indicate that histone lactoylation is site-specific and regulated by GLO2, with significant increases in specific histone marks like H3K18lac in KO cells. Chromatin accessibility analysis using ATAC-seq shows that GLO2 knockout leads to widespread changes in chromatin accessibility, with increased accessibility at specific genomic loci. These changes are associated with increased H3K18lac, suggesting that histone lactoylation plays a role in gene activation. Overall, the study highlights the importance of LGSH in regulating inflammatory signaling through histone lactoylation.