Testes-specific protease 50 (TSP50), a novelly identified oncogene, has the capacity to induce cell proliferation, cell invasion and tumor growth. Further studies indicated that CAGA-luc (an activin-responsive reporter construct) reporter activity could be significantly suppressed by TSP50 overexpression, implying that the activin signaling may participate in TSP50-mediated cell proliferation. Here, we reported that TSP50 had an inhibitory effect on activin signaling. Mechanistic studies revealed that TSP50 could interact with ActRIIA, inhibit activin typeIreceptor (ActRIB) phosphorylation, repress Smad2/3 nuclear accumulation and finally promote cell proliferation by reducing the expression of activin signal target gene p27. Additionally, we found that ActRIB activation could reverse TSP50-mediated cell proliferation and tumor growth. Furthermore, analysis of human breast cancer specimens by immunohistochemistry indicated that TSP50 expression was negatively related to p-Smad2/3 and p27 protein levels. Most importantly, breast cancer diagnosis-related indicators such as tumor size, tumor grade, estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER-2) levels, were correlated well with TSP50/p-Samd2/3 and TSP50/p27 expression status. Thus, our studies revealed a novel regulatory mechanism underlying TSP50-induced cell proliferation and provided a new favorable intervention target for the treatment of breast cancer.