Transmembrane protease serine 4 (TMPRSS4) is a novel type II transmembrane serine protease that is overexpressed in various types of human cancers and has an important function in cancer progression. However, there is a paucity of data available regarding the biological effects of TMPRSS4 on breast cancer (BC) cells and the underlying mechanisms. In this study, expression of TMPRSS4 in BC tissues was detected by immunohistochemistry. The relationship between TMPRSS4 expression and clinicopathological characteristics as well as prognosis was evaluated. The effects of TMPRSS4 on cell proliferation, migration and invasion were investigated in BC cell lines in vitro. Additionally, RT-qPCR and western blot analysis were used to determine the expressions of epithelial-mesenchymal transition (EMT) biomarkers and TMPRSS4 in BC cell lines. We found that TMPRSS4 was overexpressed in BC tissues and its expression level was closely correlated with tumor size, histological grade, lymph node metastasis, clinical stage as well as poor survival (all P<0.05) and could be recognized as an independent prognostic factor for BC patients. Overexpression of TMPRSS4 promoted the proliferation, migration and invasion of BC cells in vitro. Moreover, TMPRSS4 knockdown significantly enhanced the expression of E-cadherin and claudin-1 and inhibited the expression of vimentin and Slug, indicating suppression of EMT. Our results suggest that TMPRSS4 plays a crucial role in the progression of BC. Moreover, TMPRSS4 overexpression promoted the proliferation, invasion and migration of BC cells by possibly inducing EMT. To conclude, TMPRSS4 may be a potential therapeutic target for cancer treatment.