Orexins (orexin-A and -B) are involved in the regulation of food intake in mammals. In the barfin flounder, Verasper moseri, we previously reported that orexin-A-like-immunoreactive (ir) cell bodies are localized in the hypothalamus, which is a possible orexigenic center in fish. However, the physiological roles of orexin in the barfin flounder remain unclear. Here, we cloned prepro-orexin cDNA and examined the effects of feeding status on orexin gene expression in the barfin flounder to obtain a better insight into the roles of orexins in feeding regulation. A molecular cloning study showed that barfin flounder prepro-orexin cDNA encodes a 145 amino acid (aa) polypeptide containing orexin-A (43 aa) and orexin-B (28 aa). Prepro-orexin gene transcripts were detected in the hypothalamus, pituitary, and several peripheral organs such as the eyeball, gills, head kidney, body kidney, spleen, testis, and the skin on the eye-side of the flounder's body. Furthermore, the mean prepro-orexin mRNA expression level in the hypothalamus was significantly higher in fasted than in fed fish. These results show that fasting regulates orexin mRNA in the hypothalamus and suggest that orexin is involved in feeding regulation in barfin flounder.