Abstract Background: The purpose of this study was to assess the diagnostic and prognostic impact of preoperative serum determination of human epididymis protein 4 (sHE4), and to investigate its potential correlation with clinicopathological features and survival endpoints in endometrial cancer patients. Methods: Preoperative serum samples from 193 endometrial cancer patients and 125 women with normal endometrium were measured for sHE4 and serum CA125 (sCA125) concentrations by quantitative chemiluminescent microparticle immunoassays on the automated Architect instrument. Results: sHE4 concentrations were significantly higher in endometrial cancer patients regardless of tumour stage and grade compared with normal controls. Setting the specificity at 95%, the sensitivities in detecting endometrial cancer patients were 66% for HE4, 33% for CA125 and 64% for the combination of the two markers. High concentrations of both HE4 and CA125 significantly correlated with all clinicopathological features characterising a more aggressive tumour phenotype. In multivariate analysis, only high preoperative sHE4 concentrations, but not sCA125, were independent prognostic factors for shorter Overall Survival, Disease-Free Survival and Progression-Free Survival. Conclusions: HE4 is more sensitive and specific than CA125 in distinguishing endometrial cancer patients from women with normal endometrium, regardless of tumour stage and grade. sHE4 appears to be associated with a more aggressive tumour variant and it could be clinically useful, in identifying high-risk endometrial cancer patients, for a tailored surgical and postoperative therapy. HE4 significant correlation with decreased Overall Survival, Disease Free Survival and Progression Free Survival suggests its potential role as a novel prognostic marker for endometrial cancer.