Earlier studies identified testes-specific protease 50 (TSP50), which encodes a threonine protease, and showed that it was abnormally reactivated in many breast cancer biopsies. Further, it was shown to be negatively regulated by the p53 gene. However, little is known about the biological function of TSP50. In this study, we applied RNA interference to knockdown TSP50 gene expression in P19 murine embryonal carcinoma stem cells and tested whether this modulated the cell phenotype. The results showed that downregulation of TSP50 expression not only reduced cell proliferation, colony formation, and migration but also induced cell apoptosis. Further investigation revealed that knockdown of TSP50 resulted in greater sensitivity to doxorubicin-induced apoptosis and that activation of caspase-3 was involved in this process.