Background: To determine reference intervals for serum levels of human epididymis protein 4 (HE4) in Chinese women. Methods: In this multicenter (n = 9) study, 618 healthy women, 767 patients with non-malignant diseases, and 951 patients with malignant tumors were enrolled. Serum levels of HE4 were measured in all patients using electrochemiluminescence immunoassays. The influence of age, menopause, malignancy status and other characteristics on the levels of HE4 was evaluated using univariate and multivariate analyses. Confidence intervals (2.5-97.5 %) were determined in different populations. Results: There were significant differences in HE4 levels among groups with different ages, menopause or malignancy status. Higher levels of HE4 were detected in elder compared to younger, post- compare to pre-menopause and malignant compared to benign subjects. Multivariate analysis showed that menopause and malignancy status, as well as smoking and pelvic masses were independent factors involved in serum HE4 levels. In pre-menopause stage, the reference ranges of HE4 level were 29.30-68.79, 28.12-1284.83 and 34.75-981.91 pmol/L in healthy, benign and malignant populations, respectively. In post-menopause stage, the reference ranges are 35.96-114.43, 39.11-2208.70 and 39.40-1678.13 pmol/L for those populations. Conclusions: The present study has established the reference intervals of HE4 levels in pre- and post-menopause populations with different malignancy status.