Acquired or inherent drug resistance is the major problem in achieving successful cancer treatment. However, the mechanism(s) of pleiotropic drug resistance remains obscure. We have identified and characterized a cellular metabolic strategy that differentiates drug‐resistant cells from drug‐sensitive cells. This strategy may serve to protect drug‐resistant cells from damage caused by chemotherapeutic agents and radiation. We show that drug‐resistant cells have low mitochondrial membrane potential, use nonglucose carbon sources (fatty acids) for mitochondrial oxygen consumption when glucose becomes limited, and are protected from exogenous stress such as radiation. In addition, drug‐resistant cells express high levels of mitochondrial uncoupling protein 2 (UCP2). The discovery of this metabolic strategy potentially facilitates the design of novel therapeutic approaches to drug resistance.—Harper, M.‐E., Antoniou, A., Villalobos‐Menuey, E., Russo, A., Trauger, R., Vendemelio, George, A. M., Bartholomew, R., Carlo, D., Shaikh, A., Kupperman, J., Newell, E. W., Bespalov, I. A., Wallace, S. S., Liu, Y., Rogers, J. R., Gibbs, G. L., Leahy, J. L., Camley, R. E., Melamede, R., Newell, M. K. Characterization of a novel metabolic strategy used by drug‐resistant tumor cells. FASEB J. 16, 1550–1557 (2002)