Objective To investigate the predictive value of preoperative serum human epididymis protein 4 (HE4) and CA125 in patients with high risk of stage Ⅰ endometrial cancer (EC)and to identify the optimal cutoff values. Methods Clinical and pathological data of 231 patients with stage Ⅰ EC were included in this study. Patients were divided into high risk group (n=96) and low risk group (n=135). The preoperative serum levels of HE4 and CA125 were measured, and their correlations with clinical pathological features were analyzed. The ROC curves were generated to determine optimal cutoff values of HE4 and CA125 levels with the maximum Youden index for prediction of high risk EC. Results There were significant differences in serum levels of HE4 and CA125 between patients with different depths of myometrial invasion, with or without vascular invasion, with or without lower uterine segment involvement, with different diameters of tumor and different risk classifications of stage ⅠEC (P<0.05). There were significant differences in serum levels of HE4 between patients with different menopausal status, hypertension, pathological types, histological grading and the involvement of cervical endometrial glands (P<0.05). The preoperative serum levels of HE4 and CA125 were positive correlated (r=0.262, P<0.05). The AUC value of HE4 for diagnosing stageⅠEC was 0.794(95%CI:0.734-0.854),the cutoff value was 74 pmol/L, the sensitivity was 75.0%, specificity was 83.0%, positive predictive value was 75.8%, negative predictive value was 82.4% and the accuracy rate was 79.7%. The AUC value of CA125 for diagnosing stage Ⅰ EC was 0.696 (95%CI: 0.624-0.767), the cutoff value was 17 kU/L, the sensitivity was 56.3%, specificity was 85.9%, positive predictive value was 74.0%, negative predictive value was 73.4%and the accuracy rate was 73.6%, respectively. The AUC value of combination of both markers was 0.847 (95%CI: 0.796-0.899), the corresponding values were 95.8%, 77.0%, 74.8%, 96.3%and 84.8%, respectively. Conclusion The best cutoff values of HE4 and CA125 are 74 pmol/L and 17 kU/L for detecting high risk stageⅠEC. The combined detection is better than that of the single detection in sensitivity, negative predictive value and accuracy rate.