Fatty acid synthase (FAS) inhibition initiates selective apoptosis of cancer cells both in vivo and in vitro, which may involve malonyl-CoA metabolism. These findings have led to the exploration of malonyl-CoA decarboxylase (MCD) as a potential novel target for cancer treatment. MCD regulates the levels of cellular malonyl-CoA through the decarboxylation of malonyl-CoA to acetyl-CoA. Malonyl-CoA is both a substrate for FAS and an inhibitor of fatty acid oxidation acting as a metabolic switch between anabolic fatty acid synthesis and catabolic fatty acid oxidation. We now report that the treatment of human breast cancer (MCF7) cells with MCD small interference RNA (siRNA) reduces MCD expression and activity, reduces adenosine triphosphate levels, and is cytotoxic to MCF7 cells, but not to human fibroblasts. In addition, we synthesized a small-molecule inhibitor of MCD, 5-{(Morpholine-4-carbonyl)-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-phenyl]-amino}-pentanoic acid methyl ester (MPA). Similar to MCD siRNA, MPA inhibits MCD activity in MCF7 cells, increases cellular malonyl-CoA levels and is cytotoxic to a number of human breast cancer cell lines in vitro. Taken together, these data indicate that MCD-induced cytotoxicity is likely mediated through malonyl-CoA metabolism. These findings support the hypothesis that MCD is a potential therapeutic target for cancer therapy.