Human epididymis protein 4 (HE4) is well known to be a predictor of ovarian cancer clinically. HE4 is reported to play crucial roles in ovarian cancer progression and metastasis. The purpose of the present study was to explore its biological role and molecular mechanism in ovarian cancer. In our study, we found that expression levels of HE4 in tissues, serum and urine in ovarian cancer were upregulated compared to healthy and benign groups. HE4 expression was elevated in ovarian cancer cells. Knockdown of HE4 dampened cell proliferation and Ki67 expression, as well as enhanced apoptosis, caspase-3 activity and cleaved-caspase-3 expression. In addition, HE4 downregulation repressed invasion and migration capabilities of ovarian cancer cells. Western blot analyses showed that knockdown of HE4 reduced the levels of matrix metalloproteinases (MMP-2 and MMP-9) and inhibited epithelial to mesenchymal transition (EMT) in ovarian cancer cells. animal experiments revealed that HE4 downregulation constrained the growth of xenograft tumor. Mechanism research showed that knockdown HE4 inhibited the activity of JAK/STAT3 pathway and In conclusion, our findings reported that knockdown of HE4 suppresses aggressive cell growth and malignant progression of ovarian cancer by inhibiting the JAK/STAT3 pathway, which provides valuable insights to contribute to develop novel HE4-targeted therapies.