The plateau pika (Ochotona curzoniae) is a small mammal native to the Qinghai-Tibetan Plateau. Pikas breed in summer and become reproductively dormant in winter. Long-day stimulation (16h∶8h) induced spermatogonial differentiation and the resumption of spermatogenesis, while short-day treatment (8h∶16h) resulted in spermatogonial differentiation arrest. Melatonin content was significantly increased in short-day adapted animals and injection of melatonin inhibited the spermatogonial differentiation and development of advanced germ cells, while treatment with a melatonin receptor antagonist stimulated spermatogonial differentiation and spermatogenesis in the reproductively dormant animals. Levels of gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH) were significantly increased in reproductively active males while follicle-stimulating hormone (FSH) did not differ. Functional experiments revealed that GnRH injection stimulated germ cells development and initiation of spermatogenesis in non-breeding season. Furthermore, GnRH content in brain was decreased by melatonin administration. Together, data from the present demonstrated that the seasonal spermatogenesis of plateau pikas was directed by photoperiod-melatonin signal. These findings revealed a key mechanism underlying seasonal reproduction in Plateau Pikas and likely other animal species.