Elevated lactate levels were associated with chemoresistance across multiple cancer types. Mechanistically, lactylation of ATRIP at lysine 270 (Lac-K270) enhanced ATR-ATRIP interaction, resulting in aberrant ATR activation and subsequent checkpoint kinase 1 (CHK1) degradation via the ubiquitin-proteasome pathway. This rewired the ATR-CHK1 DDR pathway, enabling cancer cells to bypass chemotherapy-induced cell cycle arrest and survive genotoxic stress. Lac-K270 was catalyzed by alanyl-tRNA synthetase 1 (AARS1) and reversed by the deacetylase sirtuin 1 (SIRT1). Pharmacological inhibition of Lac-K270 with alaninol effectively re-sensitized chemoresistant tumors to genotoxic drugs in both in vitro and animal models.