Hepatocyte growth factor activator inhibitor type 1 (HAI-1), encoded by the serine protease inhibitor Kunitz type 1 (SPINT1) gene, is a transmembrane protease inhibitor that is believed to regulate the activities of various membrane-associated serine proteases. HAI-1 has two Kunitz-type inhibitor domains with the N-terminal Kunitz domain (KD1) responsible for inhibiting known target proteases. To analyze the roles for HAI-1 in cancer, we performed stable knockdown of HAI-1 in human cancer cell lines HAI-1 knockdown resulted in morphological changes suggesting epithelial to mesenchymal transition (EMT) in certain cell lines such as SUIT-2 pancreatic cancer and HLC-1 lung cancer cell lines with up-regulation of smad-interacting protein 1 (SIP1/ZEB2) and reduced E-cadherin expression. These changes are mediated partly by type-II transmembrane serine proteases, matriptase and TMPRSS4. Next, we examined the effect of HAI-1 knockdown on metastasis of SUIT-2 cells. In a nude mouse subcutaneous transplantation model, the tumor growth was rather slower in the knockdown SUIT-2 cells and the incidence of pulmonary metastasis was comparable between knockdown and control cells. However, the number of metastatic colonies per mouse was higher in the knockdown cells as long as metastasis arose. In an experimental pulmonary metastasis model with tail vein injection, metastatic colonization was apparently increased in the knockdown cells. One week after the injection, metastatic colonization was observed in 36% (4/11) of mice injected with HAI-1-knockdown SUIT-2, whereas none (0/11) of the control mice shows metastatic lesion. The metastasis ratios were 80% (4/5) and 40% (2/5) after two weeks, and 82% (9/11) and 45% (5/11) after four weeks, for HAI-1-knockdown and control SUIT-2 cells, respectively. Moreover, pretreatment with recombinant KD1 reduced metastasis. Therefore, the loss of HAI-1 promoted pulmonary colonization during the early phase of metastasis. In conclusion, HAI-1 has a suppressing role in cancer invasion and metastasis, and KD1 may serve as a therapeutic modality for inhibiting metastasis.