Abstract: Gonadal soma-derived factor（gsdf） is one of switchers of spermatogenesis during sexual differentiation in medaka（Oryzias latipes）, whilst gsdf knockout resulted in hyperproliferation of germ cells and male-to-female sex reversal. In this study, proteomics was performed to identify a global 60 up-regulated and 158 down-regulated differentially expressed proteins（DEPs） by the comparison of normal XX and gsdf deficient XY ovaries. GO（Gene ontology） assignment of DEPs enriched the pathway of intercellular bridging and lipid transport groups. RNA-degradation was one of gsdf targeted pathway identified by KEGG（Kyoto encyclopedia of genes and genomes） pathway enrichment. The protein levels of DDX6 significantly increased in gsdf-knockout ovaries, consisting with the increase of ddx6 mRNA levels revealed by transcriptomics analysis. The molecular structure of DDX6 was evolutionary conserved across species. Maternal ddx6 transcripts decreased significantly in early embryogenesis, but re-expressed in adult testis and ovary, indicating that DDX6 plays an important role in medaka（Oryzias latipes） gametogenesis and gonadal development. Evidence of protein-protein interaction networks and proteomics analysis, supported the view that gsdf inhibited germ cell proliferation by regulating RNA degradation（including ddx6, lsm1 and other genes）, and affected the assembly of P-bodies, and thus blocked the female differentiation of germ cells. However, the expression of vitellogenin in the ovaries with gsdf deletion was abnormally reduced, which was consistent with the phenomenon that a large number of oocytes stopped at the primary oocyte stage before vitellogenesis after knockout of gsdf, indicating that gsdf may participate in the primary folliculogenesis and oocyte development process through DDX6. Our results provide a novel insight into the mechanism of vertebrate sexual differentiation.