Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that play key roles in lipid and energy homeostasis. Paralichthys olivaceus PPARalpha (PoPPARalpha) cDNA was isolated by initial reverse transcription-polymerase chain reaction (RT-PCR) using conserved region among fish species and rapid amplification of cDNA ends (RACE). The full-length of PoPPARalpha cDNA is 2040-bp long encoding a polypeptide with 505 amino acids and containing a DNA binding domain (C4-type zinc finger) and a ligand-binding domain. PoPPARalpha was detected from 1 day post-hatch and was highly expressed in the stomach, liver, and intestine of continuously fed flounder, approximately 16 cm in size. PoPPARalpha mRNA expression was down-regulated in the kidney, stomach, and liver of the 4.5-month-old flounder after a 30 day food-deprivation period. PoPPARalpha activates the PPAR response element (PPRE)-driven reporter, and treatment with Wy14643, a PPARalpha agonist, augmented PoPPARalpha-stimulated peroxisome proliferator response element activity in HINAE and HepG2 cells. PoPPARalpha activated the expression of fatty acid beta-oxidation related genes such as carnitine palmitoyltransferase 1A, medium chain acyl-CoA dehydrogenase, and acyl-CoA oxidase 1 and inhibited the expression of sterol regulatory element binding protein and fatty acid synthase by competitively inhibiting LXR/RXR heterodimer formation. These results suggest that PoPPARalpha plays an important role in lipid metabolism of olive flounder and that it is functionally and evolutionarily conserved in olive flounder and mammals.