Lignans are a group of estrogenic compounds present in plants. Several epidemiological studies proposed that lignans may protect against breast cancer by exerting anticarcinogenic activity. Levels of enterolactone were determined in serum samples of 1,250 cases and 2,164 controls from a large population-based case–control study. We assessed the association between serum enterolactone and postmenopausal breast cancer risk using conditional logistic regression accounting for potential risk and confounding factors. Fractional polynomials were used to determine the function that best fitted the data. Moreover, we assessed heterogeneity by estrogen/progesterone/herceptin (ER/PR/HER2) status of the tumor. Additionally, a meta-analysis with seven further studies addressing enterolactone concentrations and breast cancer risk was performed. Postmenopausal breast cancer risk decreased with increasing serum enterolactone levels [highest compared to lowest quintile: [odds ratio = 0.65; 95% confidence interval (CI) 0.52–0.83,ptrend = <0.0001]. A significant inverse association for ER+/PR+ as well as ER−/PR− tumors was observed, with a significantly stronger association for ER−/PR− tumors (pheterogeneity = 0.03). The association for ER−/PR− tumors did not differ by expression of HER2 (pheterogeneity = 0.3). The meta-analysis yielded a significant reduced pooled risk estimate of: 0.66; 95% CI: 0.55–0.77) comparing the highest to the lowest quantiles of enterolactone levels. We found strong evidence for a significant inverse association between serum enterolactone and postmenopausal breast cancer risk, which was stronger for ER−PR− than for ER+PR+ tumors but not differential by further expression of HER2. The overall evidence together with other studies supports an inverse association between higher serum enterolactone levels and postmenopausal breast cancer risk.