[Objectives] Melatonin is an important neuroendocrine amine hormone that plays a critical role in regulating gonadal function and germ cell development in seasonal breeding animals. However, the regulatory pathway of melatonin affecting seasonal reproduction through hypothalamic-pituitary-gonadal (HPG) axis is still unclear. [Methods] In the present study, Plateau Pika (Ochotona curzoniae), a representative long-day animal, was used. Adult Pikas from reproductive and non-reproductive stages were captured in May and November, respectively, and assigned to four groups: reproductive male, reproductive female, non-reproductive male and non-reproductive female. Blood samples were collected from Plateau Pikas by posterior orbital venous plexus approach every 2 h within 24 h, and serum melatonin concentrations were detected by enzyme linked immunosorbent assay. Quantitative real-time PCR was used to determine relative expression levels of Mtnr1a and Mtnr1b with reference to Gapdh in hypothalamus, pituitary and gonad of Plateau Pika. The expression and location of MTNR1A and MTNR1B in pika’s gonad were confirmed by immunofluorescence staining with a germ cell specific marker DDX4 and a DNA specific dye Hoechst 33342. The concentration of melatonin and relative expression levels of Mtnr1a and Mtnr1b were compared among groups by one-way ANOVA. [Results] Results showed that serum melatonin concentration of non-reproductive male Pikas was always higher than that of reproductive males (P < 0.05), and the two groups showed distinct diurnal dynamics. Serum melatonin concentration of female pikas was lower than that of males all the time (P < 0.05), although there was no significant difference in female pikas between reproductive and non-reproductive stages (Fig. 1). The mRNA expressions of Mtnr1a and Mtnr1b were detected with similar changing pattern. There were significant seasonal differences in Mtnr1a and Mtnr1b expressions in male hypothalami and pituitaries (P < 0.05), and seasonal differences were also found in Mtnr1a and Mtnr1b expressions in female pituitaries and ovaries (P < 0.05) (Fig. 3). Immunostaining indicated that the two receptors were widely distributed in germ cells and Sertoli cells of testis. Nevertheless, MTNR1A was more extensively expressed in spermatogonia, while more expression of MTNR1B was observed in advanced germ cells at reproductive stage. In the ovary, MTNR1A was found in oocyte cytoplasm and granulosa cells preferentially. MTNR1B expression was observed in both oocyte nucleus and cytoplasm as well as in granulosa cells. Strikingly, distinct high expression of MTNR1B was found in theca cells of growing follicles (Fig. 4). [Conclusion] In conclusion, melatonin showed gender difference effects on seasonal reproduction in Plateau Pikas, implying that its control pattern is not limited to indirect regulation through HPG axis, but it also acts directly on melatonin receptors in gonad to control the fate of germ cells and somatic cells.