This research examined the expression characteristics and mechanism of the novel membrane progestin receptor-like (mPRL) in Japanese flounder Paralichthys olivaceus. In the present study, the temporal changes of mPRL mRNA expression level in isolated oocytes at various oogenesis stages were measured using quantitative real-time PCR (qRT-PCR). The mPRL transcript levels significantly increased with the oocyte development, with the peak level at stage Ⅴ. However, the transcript level decreased significantly at stage Ⅵ. These expression characteristics of mPRL showed that mPRL is a good marker for studying the oocyte maturation. The spatial expression of P. olivaceus mPRL mRNA in the ovary and brain was demonstrated using in situ hybridization. Results revealed that mPRL transcript was localized at the membrane of oocytes, and the positive signals were also observed in the brain neurons of P. olivaceus. Specifically, high-titer rabbit polyclonal antibody was generated against synthetic peptides derived from the C-terminal domain of P. olivaceus mPRL amino acid. Immunobloting analysis identified the immunoactive protein that bands in the ovary, brain, head kidney, kidney and liver of P. olivaceus. However, the expression levels of protein in ovary and brain were higher than other tissues. The protein was localized in the major reproductive associated organs including the ovary and brain using immunohistochemistry. Moreover, the cellular localization of mPRL protein was similar with that of mRNA in P. olivaceus. The intensive signals were observed at the edge of pituitary and brain, and the intensity of positive signals was found on the membrane of oocytes. This is to date the first study that examined the tissue specific localization of mPRL protein and transcript in the reproductive organs of P. olivaceus. The mPRL expression characteristics implied that mPRL regulates the reproduction of P. olivaceus through the endocrine system. Furthermore, the evidence supports a functional role of mPRL in the oocyte maturation of P. olivaceus, possibly, by acting as a mediator of progesterone.