BACKGROUND In China, the association between estrogen metabolism and breast cancer risk and the differences in metabolic pattern between breast cancer patients and controls are poorly understood. METHODS A total of 84 patients with invasive breast cancer and 47 controls with benign breast diseases were included in this study. Estrogen metabolites from their morning urine were determined by HPLC-MS/MS and evaluated in both groups, and the predictive value of each estrogen metabolite in the malignant group according to their menstrual status was analyzed. RESULTS Urinary concentration of estrogen metabolites 2-hydroxyestrone (2-OHE1), 2-hydroxyestradiol (2-OHE2), 4-hydroxyestradiol (4-OHE2), 4-methoxyestrone (4-MeOE1), and 16α-hydroxyestrone were lower in postmenopausal patients with breast cancer, compared with benign controls. In logistic regression model, breast cancer risk increased with the decline in the levels of 4-OHE2 and 4-MeOE1. In premenopausal patients, a difference in the level of 2-OHE2 was observed between both groups, and 2-OHE2 was found to have predictive value for breast cancer. Additionally, urinary 2-OHE2 level in premenopausal hormone receptor positive (HR+) patients was considerably higher compared with hormone receptor negative patients. CONCLUSIONS We found that lower urinary levels of 4-OHE2 and 4-MeOE1 had predictive value for breast cancer, and higher 2-OHE1 were associated with HR+ breast cancer in premenopausal women.