It is thought that peroxisome proliferator-activated receptor α (PPARα) is a major regulator for fatty acid metabolism. Long-chain fatty acids have been shown to induce expression of the genes related to fatty acid metabolism through PPARα. However, it is unclear whether the intensity of PPARα activation is different among various fatty acids. In this study, we compared various fatty acids in the capability of PPARα activation by differential protease sensitivity assay (DPSA), electrophoretic mobility shift assay and GAL4-PPAR chimera reporter assay in intestinal cell line, Caco-2. DPSA revealed that polyunsaturated fatty acids of 18 to 20 carbon groups with 3–5 double bonds strongly induced a PPARα conformational change. The ligand-induced changes in the sensitivity to protease corresponded to the enhancement of the binding of PPARα– RXRα heterodimer to the PPAR-response element (PPRE). The GAL4-PPAR chimera reporter assay revealed that the DNA binding-independent transactivity of PPARα was induced by various fatty acids with a wide spectrum of intensity which correlated with the conformational change of PPARα. These results suggest that PPARα has greater selectivity to certain types of polyunsaturated fatty acids, and that the ligand-induced conformational change of PPARα leads to parallel increases in both DNA binding to the PPAR-response element and the DNA bindingindependent transactivity.