Dominant negative thyroid hormone receptors (TR) show elevated expression relative to ligand binding TRs during cardiac hypertrophy. We tested the hypothesis that over-expression of a dominant negative TR alters cardiac metabolism and contractile efficiency (CE). We used mice expressing the cardioselective dominant negative TR 1 mutation, 337T. Isolated working 337T hearts and nontransgenic controls (CON) were perfused with ¹³carbon labeled free fatty acids (FFA), acetoacetate (ACAC), lactate, and glucose at physiological concentrations for 30 minutes. ¹³C NMR spectroscopy and isotopomer analyses were used to determine substrate flux and fractional contributions (Fc) of acetyl-CoA to the citric acid cycle (CAC). 337T hearts exhibited rate depression, but higher developed pressure and CE, defined as work per oxygen consumption (MVO₂). Unlabeled substrate Fc from endogenous sources was higher in 337T, but Fc -ACAC was lower. Fluxes through CAC, lactate, ACAC, and FFA were reduced in 337T. CE and Fc differences were reversed by pacing 337T to CON rates, accompanied with an increase in FFA Fc. 337T hearts lacked the ability to increase MVO₂. Decreases in protein expression for glucose transporter-4 and hexokinase-2 and increases in puryvate dehydrogenase kinase-2 and -4 suggest that these hearts are unable to increase carbohydrate oxidation in response to stress. These data show that 337T alters the metabolic phenotype in murine heart by reducing substrate flux for multiple pathways. Some of these changes are heart rate dependent, indicating that the substrate shift may represent an accommodation to altered contractile protein kinetics, which can be disrupted by pacing stress.