Gonadal soma-derived factor (gsdf) has been demonstrated as essential for testicular differentiation in medaka (Oryzias latipes). To understand the protein dynamics of Gsdf in spermatogenesis regulation, we used a His-tag “pull-down” assay coupled with shotgun LC-MS/MS to identify a group of potential interaction partners for Gsdf, which included cytoplasmic dynein light chain 2, eukaryotic polypeptide elongation factor 1 alpha (eEF1), and actin filaments in mature medaka testis. Alike the interaction with TGF-dynein critical for spermatogonial division in Drosophila melanogaster, the physical interaction of Gsdf & dynein and Gsdf & eEF1 were identified through a yeast 2-hybrid (Y2H) screening of an adult testis cDNA library using Gsdf as bait, and were verified by a paired Y2H assay. Co-immunoprecipitation of Gsdf and eEF1was defined in adult testes, as supporting the requirement of Gsdf and eEF1 interaction in testis development. Proteomics analysis and ultrastrutural observation showed that Gsdf deficiency activated eEF1-mediated protein synthesis and ribosome biogenesis, which in turn led to the differentiation of undifferentiated germ cell. Thus, our results provide a framework and new insight into the coordination of Gsdf (TGF and eEF1complex in the basic processes of germ cell proliferation, and transcriptional and translational control of sexual RNA, which may be fundamentally conserved across phyla during sex differentiation.