Lactylation of histone H3K9 (H3K9la) promotes temozolomide (TMZ) resistance in glioblastoma (GBM) through LUC7L2-mediated MLH1 intron retention. This study reveals that H3K9la is elevated in recurrent GBM and TMZ-resistant cells, and is enriched in the LUC7L2 promoter, activating its transcription. LUC7L2 mediates MLH1 intron 7 retention, reducing MLH1 expression and inhibiting the mismatch repair (MMR) pathway, thereby enhancing TMZ resistance. The anti-epileptic drug stiripentol, which crosses the blood-brain barrier and inhibits lactate dehydrogenase A/B (LDHA/B) activity, acts as a lactylation inhibitor, increasing GBM cell sensitivity to TMZ in vitro and in vivo. These findings highlight the role of lactylation in GBM TMZ resistance and suggest a potential combined therapeutic strategy using stiripentol to enhance TMZ efficacy. The study also demonstrates that LUC7L2 regulates MLH1 intron retention through RNA-binding activity, leading to NMD-dependent degradation of MLH1 mRNA. Stiripentol treatment significantly enhances TMZ sensitivity by inhibiting H3K9la and increasing MLH1 expression. The results provide new insights into the mechanisms of TMZ resistance in GBM and offer a promising approach for improving GBM treatment outcomes.Lactylation of histone H3K9 (H3K9la) promotes temozolomide (TMZ) resistance in glioblastoma (GBM) through LUC7L2-mediated MLH1 intron retention. This study reveals that H3K9la is elevated in recurrent GBM and TMZ-resistant cells, and is enriched in the LUC7L2 promoter, activating its transcription. LUC7L2 mediates MLH1 intron 7 retention, reducing MLH1 expression and inhibiting the mismatch repair (MMR) pathway, thereby enhancing TMZ resistance. The anti-epileptic drug stiripentol, which crosses the blood-brain barrier and inhibits lactate dehydrogenase A/B (LDHA/B) activity, acts as a lactylation inhibitor, increasing GBM cell sensitivity to TMZ in vitro and in vivo. These findings highlight the role of lactylation in GBM TMZ resistance and suggest a potential combined therapeutic strategy using stiripentol to enhance TMZ efficacy. The study also demonstrates that LUC7L2 regulates MLH1 intron retention through RNA-binding activity, leading to NMD-dependent degradation of MLH1 mRNA. Stiripentol treatment significantly enhances TMZ sensitivity by inhibiting H3K9la and increasing MLH1 expression. The results provide new insights into the mechanisms of TMZ resistance in GBM and offer a promising approach for improving GBM treatment outcomes.