Since deficiencies of critical nutrients and hypoxia are observed in hypovascular tumors, glycolysis alone cannot explain how cancer cells maintain their required energy levels. To study energy metabolism in cancer cells within such tumor microenvironments, we examined the NADH-fumarate reductase system, which is found in anaerobic organisms, such as parasitic helminthes. In human cancer cells cultured under tumor microenvironment-mimicking conditions, mitochondrial NADH-fumarate reductase activity increased in parallel with an increase in fumarate reductase activity, which is the reverse reaction of succinate-ubiquinone reductase and is regulated by the phosphorylation of its subunit. Pyrvinium pamoate, an anthelmintic drug, has an anticancer effect within tumor-mimicking microenvironments. We found that one of the biological mechanisms of pyrvinium is the inhibition of the NADH-fumarate reductase system. Therefore, the NADH-fumarate reductase system might be important for maintaining mitochondrial energy metabolism within the tumor microenvironments and might represent a novel target for anticancer therapies.