Objective To investigate the effect of overexpression of human epididymal protein 4 (HE4) on proliferation, invasion and tumorigenesis of endometrial carcinoma (EC) cells. Methods HE4 overexpression vector (pcDNA 3.1/Myc-His-HE4) and HE4 specific small interfering RNA (siRNA-HE4) were constructed and respectively transfected into HEC-1B (HE4 overexpression group) and Ark2 (HE4 low expression group), the two EC cell lines. HEC-1B cells transfected with empty vector pcDNA 3.1/Myc-His were used as negative control for the HE4 overexpression group. Ark2 cells transfected with nonspecific sequence siRNA were used as negative control for the HE4 low expression group. The EC cell line without transfection and normal culture served as a normal control group. After transfection, the expression levels of HE4 mRNA were detected by quantitative real-time PCR (Real-time PCR), and cell proliferation was measured by methylthiazoletrazolium (MTT) assay. Migration and invasion of cells were examined by migration and invasion experiments in vitro. The transplanted tumor mice model were constructed and the tumor volume were compared. Results Compared with the HEC-1B cells in the normal control group and negative control group, the expression level of HE4 mRNA in the HEC-1B cells increased significantly after transfection with pcDNA 3.1/Myc-His-HE4 (P<0.05), and the cell proliferation activity, transmembrane cell number increased obviously (P<0.05). Compared with the Ark2 cells in the negative control group and the normal control group, the expression of HE4 mRNA in the Ark2 cells decreased significantly after transfection with siRNA-HE4 (P<0.05), the cell proliferation activity and transmembrane cell number also significantly decreased (P<0.05). While, no significant difference between the negative control group and the normal control group was found (P> 0.05). The volume and weight of transplanted tumors in the HE4 overexpression group were significantly higher than those in the other two control groups (P<0.05). And the volume and weight of transplanted tumors in the HE4 low expression group were significantly smaller than those in the other two control groups (P<0.05). Conclusion Overexpression of HE4 could lead to the proliferation, migration and invasion of EC cells, and promote tumor formation. Downregulation of HE4 gene expression could inhibit the proliferation, migration and invasion of EC cells and inhibit the tumor growth.