Nesfatin-1, a newly discovered satiety molecule, is located in the hypothalamic nuclei, including the paraventricular nucleus (PVN) and supraoptic nucleus (SON). In this study, fine localization and regulation of nesfatin-1 neurons in the PVN and SON were investigated by immunohistochemistry of neuropeptides and c-Fos. In the PVN, 24% of nesfatin-1 neurons overlapped with oxytocin, 18% with vasopressin, 13% with CRH, and 12% with TRH neurons. In the SON, 35% of nesfatin-1 neurons overlapped with oxytocin and 28% with vasopressin. After a 48-h fast, refeeding for 2 h dramatically increased the number of nesfatin-1 neurons expressing c-Fos immunoreactivity by approximately 10 times in the PVN and 30 times in the SON, compared with the fasting controls. In the SON, refeeding also significantly increased the number of nesfatin-1-immunoreactive neurons and NUCB2 mRNA expression, compared with fasting. These results indicate that nesfatin-1 neurons in the PVN and SON highly overlap with oxytocin and vasopressin neurons and that they are activated markedly by refeeding. Feeding-activated nesfatin-1 neurons in the PVN and SON could play a role in the postprandial regulation of feeding behavior and energy homeostasis.