The study investigates the role of NBS1 lactylation in DNA repair and chemotherapy resistance. Lactate accumulation, a hallmark of cancer, promotes NBS1 lactylation at lysine 388 (K388), which is essential for the formation of the MRE11–RAD50–NBS1 (MRN) complex and the accumulation of homologous recombination (HR) repair proteins at DNA double-strand break sites. TIP60 is identified as the NBS1 lysine lactyltransferase, while HDAC3 acts as the de-lactylase. High levels of NBS1 K388 lactylation predict poor patient outcomes for neoadjuvant chemotherapy. Inhibiting lactate production, either through genetic depletion of lactate dehydrogenase A (LDHA) or using stiripentol, a lactate dehydrogenase A inhibitor, inhibited NBS1 K388 lactylation, reduced DNA repair efficacy, and overcame chemotherapy resistance. These findings identify NBS1 lactylation as a critical mechanism for genome stability and chemotherapy resistance, suggesting that inhibiting lactate production could be a promising therapeutic strategy for cancer treatment.The study investigates the role of NBS1 lactylation in DNA repair and chemotherapy resistance. Lactate accumulation, a hallmark of cancer, promotes NBS1 lactylation at lysine 388 (K388), which is essential for the formation of the MRE11–RAD50–NBS1 (MRN) complex and the accumulation of homologous recombination (HR) repair proteins at DNA double-strand break sites. TIP60 is identified as the NBS1 lysine lactyltransferase, while HDAC3 acts as the de-lactylase. High levels of NBS1 K388 lactylation predict poor patient outcomes for neoadjuvant chemotherapy. Inhibiting lactate production, either through genetic depletion of lactate dehydrogenase A (LDHA) or using stiripentol, a lactate dehydrogenase A inhibitor, inhibited NBS1 K388 lactylation, reduced DNA repair efficacy, and overcame chemotherapy resistance. These findings identify NBS1 lactylation as a critical mechanism for genome stability and chemotherapy resistance, suggesting that inhibiting lactate production could be a promising therapeutic strategy for cancer treatment.