Objective: In the current study, we investigated the mechanism relating downregulation of mitogen-activated protein kinase kinase-4 (MKK4) expression to the development of endometrial cancer. Methods: MKK4 expression in endometrial cancer was assessed by immunohistochemistry using 87 paraffin-embedded tissue specimens, and clinical data was collected via a retrospective chart review. MKK4 gene knockdown using silencing RNA and an MKK4 gene transfection system was used to assess MKK4 function in tissue samples of endometrial cancer. Results: Lower expression of MKK4 immunointensity was observed in 63.2% (55/87) of the analyzed tumors. High-grade endometrioid adenocarcinoma (G2 and G3) (p = 0.024), postmenopausal status (p = 0.018), and patient age (≧60) (p = 0.012) were significantly correlated with lower MKK4 expression. Patients with lower MKK4 expression in endometrial cancer tissues tended to have a shorter overall survival (p = 0.197). Using cell growth and anchorage-independent assays, we determined that both the growth and colony-forming ability of MKK4-transfected HEC1B cells, a line with a low endogenous expression of MKK4, were significantly reduced compared to control vector-transfected cells. Overexpression of the MKK4 gene in HEC1B cells resulted in reduced cell migration activity in a simulated wound healing assay. To confirm that MKK4 expression is related to tumor suppressor function, we used 2 independent but complementary approaches. MKK4 gene knockdown in JHEM1 cells, which overexpressed MKK4, increased proliferation activity. Additionally, the engineered expression of MKK4 in Ishikawa cells, a line with low endogenous MKK4 expression, produced a phenotype similar to that of HEC1B. Similar results were produced in tumor xenografts in nude mice. Conclusion: These results indicate that MKK4 acts as a tumor suppressor, and reduced expression of MKK4 may contribute to the development of endometrial cancer.