Pyroglutamylated RFamide peptide (QRFP), an important regulator of metabolism and energy homeostasis, has orexigenic effects. QRFP acts via a specific receptor, Gpr103. Gpr103 mRNA is expressed in the rat hypothalamic paraventricular nucleus (PVN). In the PVN, corticotropin-releasing factor (CRF), which plays a central role in regulating the stress response and is produced in response to stress, stimulates the release of adrenocorticotropic hormone from the anterior pituitary. We hypothesized that QRFP regulates CRF gene expression directly in the hypothalamus, and thus examined the direct effect of QRFP on the promoter activity and mRNA levels of CRF in hypothalamic cells. To examine these pathways, we used hypothalamic 4B cells, a homologous PVN neuronal cell line. Gpr103a and Gpr103b mRNA, and Gpr103 (a and b) proteins were expressed in the hypothalamic cells. The Gpr103 mRNA and protein levels were increased by QRFP. QRFP also stimulated CRF mRNA levels and CRF promoter activity directly in 4B cells following their transfection with the CRF promoter. The protein kinase A (PKA) and protein kinase C (PKC) pathways were involved in the QRFP-induced increases in CRF promoter activity. QRFP stimulated cAMP response element-binding protein (CREB) phosphorylation. CREB phosphorylation was inhibited by a PKC inhibitor. PKC-dependent signaling would be upstream of the CREB phosphorylation. Thus, QRFP-dependent pathways are involved in the regulation of CRF gene expression in the hypothalamus.