Abstract: Glial cell line-derived neurotrophic factor (GDNF) is a key factor mediating the self-renewal and maintenance of spermatogonial stem cells (SSCs). Our previous research indicated that there were two gdnf genes in medaka (Oryzias latipes), namely gdnfa and gdnfb, both of which were expressed in the testis and play important roles in the spermatogonial stem cell line SG3. However, the cellular expression patterns of gdnfa and gdnfb in the testis, as well as regulation of their expressions by retinoic acid (RA) and androgen, remain unclear. In this study, the cellular expression patterns of medaka gdnfa and gdnfb in the testis were detected using fluorescence in situ hybridization (FISH). Furthermore, the regulation of RA and 11-ketotestosterone (11-KT) on the expression of gdnfa and gdnfb was investigated at the tissue, cell, and molecular levels through real-time quantitative polymerase chain reaction (qRT-PCR) and transcriptional activity analyses of their 5′ upstream sequences. These results indicated that in medaka testicular sections, gdnfa was primarily expressed in somatic cells, while gdnfb was expressed in both germ cells and somatic cells. In testicular organ culture and somatic cells MTS1 derived from adult medaka testis, RA and 11-KT at different concentrations significantly down-regulated the expression of gdnfa, whereas up-regulated the expression of gdnfb. moreover, RA and 11-KT treatment reduced the luciferase activity of different truncated 5′ upstream sequences of gdnfa, but enhanced the luciferase activity of different truncated 5′ upstream sequences of gdnfb. Collectively, medaka gdnfa and gdnfb exhibit differential cellular expression patterns in the testis and are differentially regulated by RA and 11-KT at the tissue, cell, and molecular levels. This study deepens our understanding of the cellular expression patterns and regulation of medaka gdnfa and gdnfb, and lays an important foundation for further research on their biological functions.