Lactate-driven lactylation of NBS1 promotes homologous recombination (HR)-mediated DNA repair and chemotherapy resistance. This study reveals that lactate induces NBS1 K388 lactylation, which is essential for the formation of the MRE11–RAD50–NBS1 (MRN) complex and the accumulation of HR repair proteins at DNA double-strand break sites. TIP60 acts as the lysine lactyltransferase for NBS1 K388 lactylation, while HDAC3 functions as the de-lactylase. High levels of NBS1 K388 lactylation are associated with poor patient outcomes in neoadjuvant chemotherapy. Lactate reduction using genetic depletion of lactate dehydrogenase A (LDHA) or the clinical inhibitor stripentol inhibits NBS1 K388 lactylation, decreases DNA repair efficacy, and overcomes chemotherapy resistance. Lactate promotes HR-mediated DNA repair and resistance to DNA-damaging therapies, including cisplatin and ionizing radiation. Lactate deprivation disrupts DNA repair, and the clinical inhibitor stripentol enhances the efficacy of DNA-damaging treatments. NBS1 K388 lactylation is correlated with poor survival in gastric cancer patients. These findings highlight the role of NBS1 lactylation in genome stability and chemotherapy resistance, suggesting that inhibiting lactate production could be a promising therapeutic strategy for cancer.Lactate-driven lactylation of NBS1 promotes homologous recombination (HR)-mediated DNA repair and chemotherapy resistance. This study reveals that lactate induces NBS1 K388 lactylation, which is essential for the formation of the MRE11–RAD50–NBS1 (MRN) complex and the accumulation of HR repair proteins at DNA double-strand break sites. TIP60 acts as the lysine lactyltransferase for NBS1 K388 lactylation, while HDAC3 functions as the de-lactylase. High levels of NBS1 K388 lactylation are associated with poor patient outcomes in neoadjuvant chemotherapy. Lactate reduction using genetic depletion of lactate dehydrogenase A (LDHA) or the clinical inhibitor stripentol inhibits NBS1 K388 lactylation, decreases DNA repair efficacy, and overcomes chemotherapy resistance. Lactate promotes HR-mediated DNA repair and resistance to DNA-damaging therapies, including cisplatin and ionizing radiation. Lactate deprivation disrupts DNA repair, and the clinical inhibitor stripentol enhances the efficacy of DNA-damaging treatments. NBS1 K388 lactylation is correlated with poor survival in gastric cancer patients. These findings highlight the role of NBS1 lactylation in genome stability and chemotherapy resistance, suggesting that inhibiting lactate production could be a promising therapeutic strategy for cancer.