Objective:To observe the ability of self-renewal and differentiation of mouse spermatogonial stem cells(SSCs)in vitro. Methods:Mouse spermatogonial stem cell line was established stably. The capacity of SSCs self-renewal in vitro was detected via EdU Proliferation Kit; Cell proliferation was observed by Live cell Imaging System. Cell apoptosis was tested by TUNEL method. The capacity of SSCs differentiation was observed by retinoic acid (RA) induction. Results:After incubation with EdU for 2 hours,the proliferation rate was analyzed as an average of 40.75% by EdU Flow Cytometry Assay Kits. The dividing stem cells were clearly catched via short-term live cell imaging. The signal of apoptosis of SSCs was nearly undetected by TUNEL methods. The expression of genes(C-kit,Scp3 and Stra8)marking differentiation was increased after RA induction(P < 0.05,n=3). Conclusion:Similar as SSCs in vivo,SSC line in vitro also has the capacity of self-renewal and differentiation. It provides us a good platform to carry out the function of the related genes and proteins during mouse spermatogenesis.