Peroxisome proliferator-activated receptors (PPARs) play an important role in the transcriptional regulation of lipid utilization and storage in several organs, including liver and heart. Our working hypothesis is that treatment of obesity/hyperlipedemia with the PPARalpha ligand fenofibrate leads to drainage of plasma lipids by the liver, resulting in reduced myocardial lipid supply, reduced myocardial fatty acid oxidation and improved myocardial tolerance to ischemic stress. Thus, we investigated changes in substrate utilization in heart and liver, as well as post-ischemic functional recovery in hearts from diet-induced obese (DIO) mice following long-term (11-12 weeks) treatment with fenofibrate. The present study shows that DIO mice express increased plasma lipids and glucose, as well as increased myocardial fatty acid oxidation and a concomitant decrease in glucose oxidation. The lipid-lowering effect of fenofibrate was associated with increased hepatic mitochondrial and peroxisomal fatty acid oxidation, as indicated by a more than 30% increase in hepatic palmiotyl-CoA oxidation and more than a 10-fold increase in acyl-CoA oxidase (ACO) activity. In line with an adaptation to the reduced myocardial lipid supply, isolated hearts from fenofibrate-treated DIO mice showed increased glucose oxidation and decreased fatty acid oxidation, as well as reduced ACO activity. Fenofibrate treatment also prevented the diet-induced decrease in cardiac function and improved post-ischemic functional recovery. We also found that, while fenofibrate treatment markedly increased the expression of PPARalpha target genes in the liver, there were no such changes in the heart. These data demonstrate that fenofibrate results in a direct activation of PPARalpha in the liver with increased hepatic drainage of plasma lipids, while the cardiac effect of the compound most likely is secondary to its lipid-lowering effect.