CBX2 (Chromobox homolog2), as a key member of the Polycomb Group protein (PcG) family, plays an important role in gonadal development. Mice lacking cbx2 showed defects in gonads (both ovary and testis) development, and some mice have exhibited male-to-female sex reversal. But little is known about the function of cbx2 in gonadal development of fish. In this study, we used Oryzias latipes, a model organism, to investigate the expression of cbx2 during embryonic and gonadal development by real-time PCR and immunohistochemistry. Real-time PCR results showed that cbx2 was highly expressed in embryos at gastrula, neurula and organogenesis stages. In bisexual gonads, cbx2 had a relatively high expression in testis. Immunohistochemistry results revealed that cbx2 mRNA was mainly localized in spermatogonia and spermatocytes in testis. And cbx2 was also predominately observed in oocytes at stages I, II and III in ovary. In order to further study the function of cbx2, we used microinjection technology to knock out cbx2. The results showed that the expression of cbx2 was significantly decreased after injecting siRNA, and thus the expression of sox9 (SRY-related HMG box 9) was down-regulated, whereas the expression of foxl2 (Forkhead transcriptional factor 2) increased. This indicated that cbx2 was not only involved in embryonic development but also played a role in gonadal differentiation and gametogenesis in O. latipes.