Peroxisome proliferator-activated receptors (PPARα, -γ and -δ) are nuclear receptors involved in transcriptional regulations of lipid metabolism. The effect of PPARα in regulation of cardiac fatty acid oxidation has been well characterized. Whether PPARδ also independently regulates fatty acid oxidation in the heart remains unclear. In this study, we tested the hypothesis that PPARδ activates fatty acids oxidation in cardiomyocytes through transcriptional activation that are independent of PPARα. Our results first indicate that PPARδ abundantly expresses in nucleus of cardiomyocytes. Palmitate oxidation rates were significantly increased in both neonatal and adult cardiomyocytes after treatment of a PPARδ-selective ligand (GW0742). Further increases of fatty acid oxidation were evident when the treatment was applied to cardiomyocytes overexpressing a wild type PPARδ, but not a mutant PPARδ that lacks the intact carboxyl ligand-binding domain. Furthermore, genes of fatty acid oxidation enzymes were significantly upregulated in cultured rat neonatal cardiomylocytes when exposed to GW0742. GW0742 can restore partly the expression of certain key genes of fatty acid oxidation in mouse adult cardiomyocytes ioslated from PPARα knockout mice. Therefore, while active crosstalk between PPARδ and -α may exist, PPARδ regulates cardiac fatty acid oxidation in the heart at least partly independent of PPARα. We conclude that PPARδ may play a key role in cardiac energy balance and may serve as a “sensor” of fatty acid of other endogenous ligands in controlling fatty acids oxidation levels in the hearts under normal and pathological conditions.