<jats:sec> <jats:title>Introduction:</jats:title> <jats:p>Itch is an unpleasant sensation that evokes a scratching behavior which often damages the skin. Nalfurafine is a kappa opioid receptor (KOR) agonist known as an effective drug used to control the intractable itch. Mechanistically, the spinal cord is a target of nalfurafine, however, little is known about the specific sites important to the antipruritic effects of nalfurafine. Therefore, the aim of this study was an investigation to uncover the sites of action of nalfurafine in the spinal neuronal pathway of itch.</jats:p> </jats:sec> <jats:sec> <jats:title>Materials and Methods:</jats:title> <jats:p>To reveal the antipruritic action of nalfurafine in the murine spinal dorsal horn, we conducted in vivo electrophysiology, behavioral experiments, and high-sensitive in situ hybridization (ISH) using normal C57BL/6J mice.</jats:p> </jats:sec> <jats:sec> <jats:title>Results:</jats:title> <jats:p>Behavioral analyses indicated that intrathecal injection of nalfurafine reduced, but not entirely eliminated the gastrin-releasing peptide (GRP)-evoked scratching bouts. In vivo electrophysiological recordings revealed that nalfurafine administration suppressed chloroquine (CQ)-responsive dorsal horn neurons in 15.8% (3/19) of mice. In fact, only 1 of 3 nalfurafine-suppressed mice responded to GRP. ISH in 3 sections of the spinal cord showed that 24.8% (154/623) were double-positive for GRP and KOR and 13.6% (68/431) for GRP receptor (GRPR) and KOR in total KOR<jats:sup>+</jats:sup> cells. Most KOR<jats:sup>+</jats:sup> cells were negative for GRP and GRPR. Intrathecal injection of dynorphin-saporin did not change the number of scratching bouts caused by GRP. However, it reduced the number of scratching bouts evoked by intradermal injection of CQ.</jats:p> </jats:sec> <jats:sec> <jats:title>Discussion:</jats:title> <jats:p>In conclusion, our data suggest that nalfurafine targets both GRP<jats:sup>+</jats:sup> KOR<jats:sup>+</jats:sup> and GRPR<jats:sup>+</jats:sup> KOR<jats:sup>+</jats:sup> cells which are present in a 2:1 ratio and suppresses CQ-induced itch in the spinal dorsal horn. These findings suggest that GRP<jats:sup>+</jats:sup> KOR<jats:sup>-</jats:sup> or GRPR<jats:sup>+</jats:sup> KOR<jats:sup>-</jats:sup> cells may function as interneurons in the spinal neuronal pathway of itch.</jats:p> </jats:sec>