TMPRSS4 is a novel type II transmembrane serine protease found at the cell surface that is highly expressed in pancreatic, colon and gastric cancer tissues. However, the biological functions of TMPRSS4 in cancer are unknown. Here we show, using reverse transcription PCR, that TMPRSS4 is highly elevated in lung cancer tissues compared with normal tissues and is also broadly expressed in a variety of human cancer cell lines. Knockdown of TMPRSS4 by small interfering RNA treatment in lung and colon cancer cell lines was associated with reduction of cell invasion and cell-matrix adhesion as well as modulation of cell proliferation. Conversely, the invasiveness, motility and adhesiveness of SW480 colon carcinoma cells were significantly enhanced by TMPRSS4 overexpression. Furthermore, overexpression of TMPRSS4 induced loss of E-cadherin-mediated cell-cell adhesion, concomitant with the induction of SIP1/ZEB2, an E-cadherin transcriptional repressor, and led to epithelial mesenchymal transition events, including morphological changes, actin reorganization and upregulation of mesenchymal markers. TMPRSS4-overexpressing cells also displayed markedly increased metastasis to the liver in nude mice upon intrasplenic injection. Taken together, these studies suggest that TMPRSS4 controls the invasive and metastatic potential of human cancer cells by facilitating an epithelial-mesenchymal transition; TMPRSS4 may be a potential therapeutic target for cancer treatment.