Japanese flounder Paralichthys olivaceus has an essential requirement for long-chain polyunsaturated fatty acids (LC-PUFA), particularly docosahexaenoic acid and eicosapentaenoic acid, but the enzymes involved in LC-PUFA biosynthesis are thought to be absent or to have low activity. Teleost fish, in particular, have quite diversified substrate preference of these enzymes even among closely related species, implying that each species could have different LC-PUFA biosynthetic capabilities. Therefore, in the present study, we characterized Japanese flounder fatty acid desaturase 2 (Fads2) and elongation of very long-chain fatty acids protein 5 (Elovl5) in order to precisely characterize the LC-PUFA biosynthesis pathway. Fads2 has Δ6 and Δ8 desaturase activity and Elovl5 has elongase activity toward C18 and C20 PUFA, suggesting that Japanese flounder is capable of synthesizing 20:4n-3 and 20:3n-6 from 18:3n-3 and 18:2n-6, respectively. Expression analysis showed that the fads2 was highly expressed in the brain and eye, while the elovl5 was highly expressed in the eye and pyloric caeca. This information will be beneficial for developing an ideal feed to support the aquaculture of Japanese flounder.