Estrogens increase breast cancer risk through estrogen receptor (ER)-mediated pathway activation. It is unclear whether a broader assessment of plasma compounds that lead to ER activation would be more strongly related to risk than measurement of individual estrogens. A prospective nested case-control study was conducted among postmenopausal women in the Nurses' Health Study, that included 371 cases with blood samples collected prior to breast cancer diagnosis and 731 matched controls. Total estrogen pathway activity (EA) was assessed via a luciferase reporter assay using plasma-treated T47D-Kbluc (ATCC) human breast cancer cells. We also assessed the contribution of EA to risk, independent of circulating estrone, estradiol, and estrone sulfate concentrations. Multivariable ORs and 95% confidence intervals (CI) were calculated using conditional logistic regression adjusting for breast cancer risk factors. Women in the highest, versus lowest EA quartile had an 86% increased risk of invasive breast cancer (ORQ4vsQ1, 1.86; 95% CI = 1.16-2.97). After accounting for estradiol only, a weaker association was observed (ORQ4vsQ1, 1.27; 95% CI = 0.75-2.17). No association was observed after accounting for all three estrogens (ORQ4vsQ1, 1.01; 95% CI = 0.56-1.84). A positive association between EA and breast cancer risk was observed. However, the association was substantially attenuated after accounting for levels of other estrogens. Our study provides a first detailed assessment of a breast cancer cell line-based EA assay and postmenopausal breast cancer risk.