Hepatocyte growth factor activator inhibitors (HAI-1 and HAI-2) are Kunitz-type serine protease inhibitors that have a broad inhibitory spectrum against serine proteases. This is the first study to investigate the role of HAI-1 and HAI-2 in endometrial cancer. We investigated the biological functions of HAI-1 and HAI-2 using KLE and HEC-251 endometrial cancer cell lines, thus HAI-1 and HAI-2 were examined in uterine normal endometrium, endometrial hyperplasia and cancer specimens by immunohistochemistry. HAI-1 and HAI-2 showed potential inhibitory effects on cell proliferation, migration and cellular invasion by reduction of matriptase and hepsin expression. This in turn led to an increase in the levels of E-cadherin and Slug, and a reduction in the levels of Vimentin, SIP1, Snail and Twist, and hence ER and PR signal transduction in endometrial cancer cells. The levels of HAI-1 and HAI-2 expression were significantly decreased in endometrial cancer specimens relative to the corresponding normal endometrium specimens. Low HAI-1 and HAI-2 expression was a significant predictor for a poor prognosis compared with high HAI-1 and HAI-2 expression. These findings indicate that HAI-1 and HAI-2 could be considered as therapeutic targets and used as favorable prognosis markers for endometrial cancer.