Abstract: Fish cell lines hold significant value in various fields such as resource conservation, genetic breeding, disease control, and environmental toxicological assessment. The establishment of gonadal cell lines provides critical in vitro models for elucidating sex determination mechanisms and gonad-related genes function regulations. In this study, we successfully established Japanese eel (Anguilla japonica) ovarian tissue cell line (AJOTCL) and testicular tissue cell line (AJTTCL) via optimizing L-15 medium supplemented with 15% fetal bovine serum (FBS), growth factors, A. japonica serum, and embryonic extracts, combined with tissue explant culture. Both cell lines were stably passaged to 120 and 100 generations, respectively, with cell survival rates exceeding 90% after cryopreservation and recovery. Morphologically, the cells predominantly exhibited fibroblast-like characteristics (spindle, prism-shaped, polygonal, or irregular shapes) with whorl-like arrangements, while AJOTCL displayed oval morphology at high density. Growth characteristic analysis revealed that medium containing 15%-20% FBS, culture temperature of 26 ℃, and a 1∶2 subculturing ratio significantly promoted proliferation capacity of AJOTCL and AJTTCL. Gene expression analysis showed that AJOTCL highly expressed ovarian-specific genes foxl2 and sox3 (similar to ovarian tissue), while AJTTCL highly expressed testicular-specific genes dmrt1 and amh (similar to testicular tissue), with both cell lines showing weak expression of the germ cell marker gene nanog or dnd, validating that both cell lines were predominantly composed of somatic cells. Therefore, they were designated as A. japonica ovarian tissue cell line and A. japonica testicular tissue cell line, respectively. Electroporation-mediated successfully transfected the exogenous gene pEGFP-N1 plasmid into both cell lines with high efficiency (≥60%), validating their applicability for genetic manipulation. Gene function validation indicated that overexpression of nanog, oct4, and lin28a/b activated downstream pathways thereby promoting cell proliferation, whereas nanog knockdown resulted in significant functional inhibition leading to cell senescence. These results demonstrate that both cell lines are suitable for gene function research. In conclusion, the gonadal somatic cell lines, AJOTCL and AJTTCL, established in this study possess stable growth characteristics and efficient gene manipulation capabilities, providing reliable in vitro models for in-depth investigation of somatic-germ cell interactions, sex determination mechanisms, and reproductive regulation.