Previous studies have shown that receptor transporter protein 4 (RTP4), one of the receptor chaperone proteins, contributes to maturation and membrane trafficking of novel opioid receptor heterodimers consisting of mu (MOPr) and delta (DOPr) opioid receptors (MOPr-DOPr). Although MOPr-DOPr is shown to contribute to the mechanism of development of antinociceptive tolerance to morphine, the role of RTP4 in such mechanism has not been elucidated yet. Since rtp4 will be upregulated by repeated administration of morphine especially in the hypothalamus, here we determined the effect of knockdown of RTP4 in the selective brain region on the development of antinociceptive tolerance to morphine by using Rtp4^flox/flox mice. In this study, Rtp4^flox/flox mice were generated and infected with AAV expressing Cre recombinase. Knockdown of rtp4 levels in hypothalamus partially but significantly suppressed the development of morphine tolerance. In addition, we found that the induction of rtp4 gene by MOPr-activation was reversed by inhibitors of Gi and MAPK pathways in neuronal cells. These results indicate that RTP4 in hypothalamus partly but significantly contributes to the mechanism of development of morphine tolerance after repeated administration of morphine. Also the upregulation of rtp4 by morphine may be mediated by MAPK pathways.