This study investigates the role of histone H3K9 lactylation (H3K9la) in temozolomide (TMZ) resistance in glioblastoma (GBM). The authors found that H3K9la is upregulated in recurrent GBM tissues and TMZ-resistant cells, and it is primarily concentrated in histone H3K9. Multi-omics analysis, including CUT&Tag, SLAM-seq, and RNA-seq, revealed that H3K9la is enriched in the LUC7L2 promoter and activates LUC7L2 transcription, which in turn mediates intron 7 retention of MLH1, reducing MLH1 expression and inhibiting mismatch repair (MMR). This results in TMZ resistance in GBM. Notably, the anti-epileptic drug stiripentol, which can cross the blood-brain barrier and inhibit lactate dehydrogenase A/B (LDHA/B) activity, acts as a lactylation inhibitor and enhances the sensitivity of GBM cells to TMZ both in vitro and in vivo. These findings provide insights into the mechanism of lactylation in TMZ resistance and suggest a potential combined therapeutic strategy for clinical GBM treatment.This study investigates the role of histone H3K9 lactylation (H3K9la) in temozolomide (TMZ) resistance in glioblastoma (GBM). The authors found that H3K9la is upregulated in recurrent GBM tissues and TMZ-resistant cells, and it is primarily concentrated in histone H3K9. Multi-omics analysis, including CUT&Tag, SLAM-seq, and RNA-seq, revealed that H3K9la is enriched in the LUC7L2 promoter and activates LUC7L2 transcription, which in turn mediates intron 7 retention of MLH1, reducing MLH1 expression and inhibiting mismatch repair (MMR). This results in TMZ resistance in GBM. Notably, the anti-epileptic drug stiripentol, which can cross the blood-brain barrier and inhibit lactate dehydrogenase A/B (LDHA/B) activity, acts as a lactylation inhibitor and enhances the sensitivity of GBM cells to TMZ both in vitro and in vivo. These findings provide insights into the mechanism of lactylation in TMZ resistance and suggest a potential combined therapeutic strategy for clinical GBM treatment.