Lactate derived from aerobic glycolysis is crucial for DNA damage repair and chemoresistance. Nevertheless, it is frequently noted that cancer cells depend on glutaminolysis to replenish essential metabolites. Whether and how glutaminolysis might enhance lactate production and facilitate DNA repair in cancer cells remains unknown. Here, it is shown that malate enzyme 2 (ME2), which metabolizes glutamine-derived malate to pyruvate, contributes to lactate production and chemotherapy resistance in ovarian cancer. Mechanistically, chemotherapy reduces the expression of glucose transporters and impairs glucose uptake in cancer cells. The resultant decrease in intracellular glucose levels triggers the acetylation of ME2 at lysine 156 by ACAT1, which in turn potentiates ME2 enzyme activity and facilitates lactate production from glutamine. ME2-derived lactate contributes to the development of acquired chemoresistance in cancer cells subjected to prolonged chemotherapy, primarily by facilitating the lactylation of proteins involved in homologous recombination repair. Targeting ACAT1 to inhibit ME2 acetylation effectively reduced chemoresistance in both in vitro and in vivo models. These findings underscore the significance of acetylated ME2-mediated lactate production from glutamine in chemoresistance, particularly under conditions of reduced intracellular glucose within cancer cell, thereby complementing the Warburg effect and offering new perspectives on the metabolic links to chemotherapy resistance.