Abstract Background Estrogen metabolite concentrations of 2-hydroxyestrone (2-OHE1) and 16-hydroxyestrone (16-OHE1) may be associated with breast carcinogenesis. However, no study has investigated their possible impact on mortality after breast cancer. Methods This population-based study was initiated in 1996-1997 with spot urine samples obtained shortly after diagnosis (mean=96 days) from 683 women newly diagnosed with first primary breast cancer and 434 age-matched women without breast cancer. We measured urinary concentrations of 2-OHE1 and 16-OHE1 using an enzyme linked immuno-assay. Vital status was determined via the National Death Index (N = 244 deaths after a median of 17.7 years of follow-up). We used multivariable-adjusted Cox proportional hazards to estimate hazard ratios (HRs) and 95% confidence intervals (95%CIs) for the estrogen metabolites-mortality association. We evaluated effect modification using likelihood ratio tests. All statistical tests were two-sided. Results Urinary concentrations of the 2-OHE1/16-OHE1 ratio (>median of 1.8 vs. ≤median) were inversely associated with all-cause mortality (HR = 0.74, 95%CI=0.56-0.98) among women with breast cancer. Reduced hazard was also observed for breast cancer mortality (HR = 0.73, 95%CI=0.45-1.17) and cardiovascular diseases mortality (HR = 0.76, 95%CI=0.47-1.23), although the 95%CIs included the null. Similar findings were also observed for women without breast cancer. The association with all-cause mortality was more pronounced among breast cancer participants who began chemotherapy before urine collection (N = 118, HR = 0.42, 95%CI=0.22-0.81) than among those who had not (N = 559, HR = 0.98, 95%CI=0.72-1.34, pinteraction=0.008. Conclusions The urinary 2-OHE1/16-OHE1 ratio may be inversely associated with long-term all-cause mortality, which may depend on cancer treatment status at the time of urine collection.