Fatty acids are the primary fuel for the heart, and are ligands for peroxisome proliferator activated receptors (PPARs), which regulates expression of genes encoding proteins involved in fatty acid metabolism. Saturated fatty acids, particularly palmitate, can be converted to the pro-apoptotic lipid intermediate, ceramide. This study assessed cardiac function, expression of PPAR regulated genes, and cardiomyocyte apoptosis in rats following eight weeks on either a low fat diet (NC; 10% fat calories), or high fat diets comprised mainly of either saturated (SAT) or unsaturated fatty acids (UNSAT) (60% fat calories) (n=10/group). The SAT group had lower plasma insulin and leptin concentrations compared to the NC or UNSAT groups. Cardiac function and mass, and body mass were not different. Cardiac triglyceride content was increased in the SAT and UNSAT groups compared to NC (p<0.05), however ceramide content was higher in SAT compared to UNSAT (2.9±0.2 vs. 1.4±0.2 nmol/g^-1; p<0.05), while the NC group was intermediate (2.3±-0.3 nmol/g^-1). The number of apoptotic myocytes, assessed by TUNEL staining, was higher in SAT compared to UNSAT (0.28±0.05 vs. 0.17±0.04 apoptotic cells/1000 nuclei; p<0.04), and was positively correlated to ceramide content (P<0.02). Both high fat diets increased the myocardial mRNA expresssion of the PPAR regulated genes encoding uncoupling protein 3 and pyruvate dehydrogenase kinase 4, but only the SAT diet upregulated medium chain acyl-CoA dehydrogenase. In conclusion, dietary fatty acid composition affects cardiac ceramide accumulation, cardiomyocyte apoptosis, and expression of PPAR regulated genes independent of cardiac mass or function.