In humans, skeletal muscle is a major site of peroxisome proliferator–activated receptor-α (PPAR-α) expression, but its function in this tissue is unclear. We investigated the role of hPPAR-α in regulating muscle lipid utilization by studying the effects of a highly selective PPAR-α agonist, GW7647, on [¹⁴C]oleate metabolism and gene expression in primary human skeletal muscle cells. Robust induction of PPAR-α protein expression occurred during muscle cell differentiation and corresponded with differentiation-dependent increases in oleate oxidation. In mature myotubes, 48-h treatment with 10–1,000 nmol/l GW7647 increased oleate oxidation dose-dependently, up to threefold. Additionally, GW7647 decreased oleate esterification into myotube triacylglycerol (TAG), up to 45%. This effect was not abolished by etomoxir, a potent inhibitor of β-oxidation, indicating that PPAR-α–mediated TAG depletion does not depend on reciprocal changes in fatty acid catabolism. Consistent with its metabolic actions, GW7647 induced mRNA expression of mitochondrial enzymes that promote fatty acid catabolism; carnitine palmityltransferase 1 and malonyl-CoA decarboxylase increased ∼2-fold, whereas pyruvate dehydrogenase kinase 4 increased 45-fold. Expression of several genes that regulate glycerolipid synthesis was not changed by GW7647 treatment, implicating involvement of other targets to explain the TAG-depleting effect of the compound. These results demonstrate a role for hPPAR-α in regulating muscle lipid homeostasis.