Background: Human epididymis protein 4 (HE4) is described as a useful new biomarker in ovarian cancer. As HE4 is neither tumor nor organ specific, we intensively investigated the occurrence of this protein in female and male patients with various benign and malignant diseases in order to avoid misinterpretation and to identify potential additional clinical relevance. Methods: We retrospectively investigated HE4 (ARCHITECT (R), Abbott Diagnostics, US) in the sera of 205 healthy individuals, 654 patients with benign disorders and 720 patients with cancer before initial treatment. Results: The lowest concentrations of HE4 were observed in healthy men (median 26.2 pmol/L) followed by healthy women (median 40.4 pmol/L). In benign diseases, highest HE4 concentrations were seen in both women and men with renal failure (women, median 1041 pmol/L; men, median 1368 pmol/L). In women, the highest HE4 levels in malignant diseases were observed in ovarian cancer (median 242 pmol/l), whereas the highest HE4 concentrations in men occurred in lung cancer (median 89.2 pmol/L). The area under the curve (AUC) of HE4 in women was highest in ovarian cancer and borderline tumors as compared to benign gynecological disorders (88.9%), with a sensitivity of 67.4% at 95% specificity. Also, significantly elevated concentrations of HE4 with reference to the respective group of benign diseases were observed in uterus corpus and breast cancer as well as in lung cancer for men and women. Conclusions: HE4 has the highest relevance in ovarian cancer but can be elevated in a variety of benign and malignant diseases.