The P-element induced wimpy testis (Piwi) proteins, which are associated with PIWI-interacting RNAs (piRNAs), play important roles in meiosis, germ cell division, and germline maintenance. In this study, we identified and characterized theParalich- thys olivaceus piwil2 gene, a constituent factor of the piRNA pathways involved in the biogenesis of reproductive development. The biological analysis indicated thatpiwil2, which contains PAZ and PIWI domains, was highly conserved between teleosts and tetrapods. Thepiwil2distribution profile in different tissues confirmed a sexually dimorphic expression pattern, with a higher expres- sion level in testis.In situ hybridization demonstrated thatpiwil2 was expressed in the oogonia and oocytes of the ovaries as well as in the Sertoli cells and spermatocytes of the testes. Genepiwil2showed a maternally inherited expression pattern during embryonic development, and was highly expressed during the early embryonic development. Different luciferase reporters were constructed to determine the transcriptional regulatory mechanisms ofpiwil2. Thepiwil2 core promoter region was located at?360bp to?60bp. Furthermore, some representative sex hormones, including human chorionic gonadotropin, 17α-methyltestosterone, and estradiol-17β had distinct regulatory effects onpiwil2. In a summery, these results indicate thatpiwil2, regulated by sex hormones and transcrip- tional elements, has vital functions in the reproductive cycle and gonadal development.