Reprogrammed glucose metabolism is one of the hallmarks of cancer, and increased expression of key glycolytic enzymes, such as pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA), has been associated with poor prognosis in various malignancies. Targeting these enzymes could attenuate aerobic glycolysis and inhibit tumor proliferation. We investigated whether the PKM2 activator, TEPP-46, and the LDHA inhibitor, FX-11, can be combined to inhibit in vitro and in vivo tumor growth in preclinical models of pancreatic cancer. We assessed PKM2 and LDHA expression, enzyme activity, and cell proliferation rate after treatment with TEPP-46, FX-11, or a combination of both. Efficacy was validated in vivo by evaluating tumor growth, PK and LDHA activity in plasma and tumors, and PKM2, LDHA, and Ki-67 expression in tumor tissues following treatment. Dual therapy synergistically inhibited pancreatic cancer cell proliferation and significantly delayed tumor growth in vivo without apparent toxicity. Treatment with TEPP-46 and FX-11 resulted in increased PK and reduced LDHA enzyme activity in plasma and tumor tissues and decreased PKM2 and LDHA expression in tumors, which was reflected by a decrease in tumor volume and proliferation. The targeting of glycolytic enzymes such as PKM2 and LDHA represents a promising therapeutic approach for the treatment of pancreatic cancer.