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The origin and evolution of molecular mechanisms underlying the self-renewal and differentiation of spermatogonial stem cells (SSCs) are fundamental questions in stem cell biology as well as reproduction medicine. In mammals, glial cell line-derived neurotrophic factor (GDNF) is crucial for SSC self-renewal and maintenance. However, in nonmammals, the role of Gdnf in SSCs still remains unknown. In this study, we report that the two GDNF homologs from medaka fish (Oryzias latipes), namely OlGdnfa and OlGdnfb, can promote proliferation activity and retain the spermatogonial property of SG3, a spermatogonial cell line derived from adult medaka showing the intrinsic property of SSCs by self-renewal and differentiation potential during 2 years of culture. Cloning and sequencing led to the identification of two cDNA sequences as Olgdnfa and Olgdnfb, which are 780-nt and 744-nt in length for 253 and 245 amino acid residues, respectively. Both are homologs of mammalian GDNF and share over 45% identity with the other known vertebrate homologs. Importantly, in a well-defined condition, the recombinant proteins, OlGdnfa and OlGdnfb, can significantly promote the proliferative activity of SG3 cells and retain the spermatogonial gene expression pattern and alkaline phosphatase activity. Meanwhile, both of the two recombinant proteins can upregulate the mRNA expression level of bcl6b, one of the prominent GDNF-regulated genes involved in SSC self-renewal and maintenance in mammals. Taken together, our findings suggest that just like the mammalian counterpart, the nonmammalian Gdnfs might mediate the self-renewal and maintenance of SSCs; moreover, Bcl6b might be a conserved regulator in SSC self-renewal across vertebrate taxa. This study extends our knowledge of GDNF functions in SSC biology during evolution.