Obestatin is a novel peptide encoded by the ghrelin precursor gene; however, its effects on gastrointestinal motility remain controversial. Here we have examined the effects of obestatin on fed and fasted motor activities in the stomach and duodenum of freely moving conscious rats. We examined the effects of intravenous (IV) injection of obestatin on the percentage motor index (%MI) and phase III-like contractions in the antrum and duodenum. The brain mechanism mediating the action of obestatin on gastroduodenal motility and the involvement of vagal afferent pathway were also examined. Between 30 and 90 min after IV injection, obestatin decreased the %MI in the antrum and prolonged the time taken to return to fasted motility in the duodenum in fed rats given 3 g of chow after 18 h of fasting. Immunohistochemical analysis demonstrated that corticotropin-releasing factor- and urocortin-2-containing neurons in the paraventricular nucleus in the hypothalamus were activated by IV injection of obestatin. Intracerebroventricular injection of CRF type 1 and type 2 receptor antagonists prevented the effects of obestatin on gastroduodenal motility. Capsaicin treatment blocked the effects of obestatin on duodenal motility but not on antral motility. Obestatin failed to antagonize ghrelin-induced stimulation of gastroduodenal motility. These results suggest that, in the fed state, obestatin inhibits motor activity in the antrum and duodenum and that CRF type 1 and type 2 receptors in the brain might be involved in these effects of obestatin on gastroduodenal motility.