Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays a central role in glycolysis. Because cancer cells rely on aerobic glycolysis rather than oxidative phosphorylation, GAPDH-depleting agents have a therapeutic potential to impede cancer cell proliferation. Knockdown of GAPDH by RNA interference induced the accelerated senescent phenotype in A549 cells, suggesting that GAPDH is a potential molecular target for combination chemotherapy. The cytotoxic effects of a panel of anticancer drugs, 5-fluorouracil, 5-fluorouridine, 5-fluorodeoxyuridine, 6-thioguanine, cytarabine, fludarabine, cladribine, clofarabine, 2-chloroadenosine, and doxorubicin, were assessed in GAPDH-depleted A549 cells using a cell proliferation assay. GAPDH-depleted A549 cells, when compared with control cells, exhibited increased chemoresistance to several antimetabolite agents including cytarabine [inhibitory concentration 50 (IC50) 1.7±0.3 vs. 0.03±0.02 μmol/l], 2-chloroadenosine (IC50 7.1±1.8 vs. 1.5±0.6 μmol/l), 6-thioguanine (IC50 7.5±1.6 vs. 1.4±0.5 μmol/l), 5-fluorouracil (IC50 13.2±2.5 vs. 3.0±0.7 μmol/l), and 5-fluorodeoxyuridine (IC50 >100 vs. 3.7±0.9 μmol/l), which we designated as group A agents. In contrast, GAPDH-deficient and GAPDH-proficient cells were equally sensitive to group B agents including doxorubicin (IC50 0.05±0.02 vs. 0.04±0.02 μmol/l), fludarabine (IC50 18.5±2.3 vs. 15.7±2.8 μmol/l), 5-fluorouridine (IC50 0.1±0.03 vs. 0.1±0.03 μmol/l), clofarabine (IC50 0.7±0.3 vs. 0.5±0.3 μmol/l), and cladribine (IC50 0.5±0.1 vs. 0.5±0.2 μmol/l). After treatment with group B agents at concentrations equivalent to 7–10-fold the IC50 value, the fraction of apoptotic cells in GAPDH-depleted, senescent A549 cells was similar to that in GAPDH-proficient cells. Our study identified the antimetabolite drugs active in senescent cells that can be used in combination with GAPDH inhibitors in cancer treatment. GAPDH-targeted combination therapy is a novel strategy to control the proliferation of tumor cells.