Citrus fruit compounds have various activities that improve pathological conditions in many tissues. In this study, we examined the effect of auraptene contained mainly in the peel of citrus on peroxisome proliferator-activated receptor-α (PPARα) activation. To examine effects of auraptene on the PPARα activation in hepatocytes, PPAR ligand assay system was developed using HepG2 hepatocytes, in which the endogenous PPARα expression level is very low. In the PPAR ligand assay, the addition of auraptene showed significant effects on the transactivation of GAL4/PPARα chimera proteins in a dose-dependent manner. Actually, treatment with auraptene induced the up-regulation of PPAR target genes, such as acyl-CoA oxidase (ACO), carnitine-palmitoyl transferase 1A (CPT1A) and acyl-CoA synthetase (ACS), in PPARα-expressing HepG2 hepatocytes. The regulation of gene expression was dependent on PPARα because mock-transfected HepG2 hepatocytes showed no regulation. The up-regulation of PPAR target gene expression by auraptene was sufficient to enhance oleic acid uptake into PPARα-expressing HepG2 hepatocytes. These results indicate that auraptene acts as a PPARα agonist in hepatocytes and that auraptene may improve lipid abnormality through PPARα activation in the liver.