Although it had been considered that pancreatic β-cells are the major source of circulating hormonal insulin, the evidence has mounted that insulin can also be synthesized in the diverse brain regions including cerebral cortex, striatum, olfactory bulb, hippocampus, and hypothalamus. Our previous study showed that acute Wnt3a injection into the third ventricle (3V) upregulates insulin gene expression in the adult mouse hypothalamus. Nevertheless, little is known which hypothalamic regions are involved in insulin production and its release. In the present study, we found a novel neuronal population expressing brain-derived insulin (BDI) in the paraventricular nucleus of the hypothalamus (PVN) by using immunolabeling and in situ hybridization. PVN BDI-expressing neurons project their axon terminals into the external zone of the median eminence (ME). Interestingly, approximately 85% of PVN BDI-expressing neurons co-express corticotrophin-releasing hormone (CRH). Furthermore, these neurons co-transport BDI and CRH into the ME. Taken together, we first report that BDI is expressed in the PVN neurons projecting to the ME.