Stearoyl-CoA desaturase (SCD) is a lipogenic enzyme, that catalyzes the synthesis of monounsaturated fatty acids. SCD1 deficiency activates metabolic pathways that promote fatty acid β-oxidation and decrease lipogenesis in liver. In the present study, we show that fatty acid (FA) transport and oxidation are decreased, whereas glucose uptake and oxidation are increased in the heart of SCD1-/- mice. Protein levels of FA transport proteins such as FAT/CD36 and FATP and activity of carnitine palmitoyltransferase 1, the rate-limiting enzyme for mitochondrial fat oxidation, were significantly lower in the heart of SCD1-/- mice compared to SCD1+/+ mice. Consequently, the rate of palmitoyl-CoA oxidation was significantly decreased in the heart of SCD1-/- mice. mRNA levels of PPAR, a key transcription factor controlling genes of FA oxidation, were significantly reduced in SCD1-/- mice. Phosphorylation of insulin receptor substrate-1 (IRS-1) as well as the association of p85 subunit of PI3-kinase with IRS-1 were significantly higher under both basal and insulin stimulated conditions in SCD1-/- hearts. This increased insulin sensitivity translated to a 1.8-fold greater 2-deoxyglucose uptake and 2-fold higher rate of glucose oxidation in the myocardium, compared to SCD1+/+ counterparts. The results suggest that SCD1 deficiency causes a shift in cardiac substrate utilization from FA to glucose by up-regulating insulin signaling, decreasing FA availability, and reducing expression of FA oxidation genes in the heart. This increase in cardiac insulin sensitivity and glucose utilization due to SCD1 deficiency could prove therapeutic in pathological conditions such as obesity that are characterized by skewed cardiac substrate utilization.