Studies have shown in mammals that retinoic acid (RA) is a key molecule mediating the initiation of meiosis, and can directly induce meiosis in cultured spermatogonial stem cells (SSCs) and express meiosis specific molecule Scp3. However, although studies have shown that RA plays an important role in testis differentiation and development and meiosis initiation in fish, its role in SSC proliferation and differentiation and whether it can directly mediate SSC meiosis remain unclear. In this study, the three-fluorescence reported plasmid pGRY, which contains three promoters, i.e., elongation factor 1α (ef1α) promoter driven histone H2B-green fluorescent protein (H2B-GFP), scp3 promoter driven puromycin-red fluorescent protein (puro-RFP), and protamine promoter driven yellow fluorescent protein (YFP), was used to transfect SG3 to obtain stable cell line SG3-pGRY. Then the effect of RA signal on the proliferation and differentiation of SG3-pGRY was investigated. The red fluorescent of SG3-pGRY could indeed reflect the expression of endogenous Scp3. Moreover, the SG3-pGRY retained stem property such as strong alkaline phosphatase activity and differentiation potential, indicating that it can be used to monitor the state of cell differentiation. With the condition of 2D culture, that is to say, the cells are sub-cultured when the cell growth density is about 90% in the cell culture plate, RA could inhibit cell proliferation, while Rar (α, β and γ) pan-antagonist BMS493 could promote cell proliferation. Furthermore, the results of real-time PCR showed that RA could significantly down-regulate the expression of pluripotency related genes pou5f3 and klf4 and had no significant effect on the expression of differentiated related genes including ckit, scp3, spo11, rec8a, rec8b except dazl. With the condition of 3D, obvious red fluorescence was observed in RA, BMS493 and control group after 48 h incubation. Compared with 2D conditions, the expressions of differentiation related genes were up-regulated compared with those in 2D culture for 48 h. In addition, the expressions of differentiation related genes in RA group were higher than the control group, whilst the BMS493 group was lower than control group. Taken together, RA signal could inhibit the proliferation and promote the differentiation of SSCs, but it is not essential to induce cell meiosis. This study not only provides a good research model for the study of fish SSC differentiation, but also promotes our in-depth understanding of the role of RA signal in fish SSC proliferation and differentiation.