The estrogen levels of Asian women are different from those of Western women, and this could affect estrogen receptor (ER) bioactivity and breast cancer risk. We conducted a case–control study in 169 postmenopausal breast cancer cases and 426 matched controls nested within a population-based prospective cohort study, the Singapore Chinese Health Study, to evaluate the serum levels of estrogens and their receptor (ERα and ERβ)-mediated estrogenic activities in relation to breast cancer risk. Breast cancer cases had higher levels of estrogens and ER-mediated bioactivities in baseline serum than the controls. Compared with those in the lowest quartile, women in the highest quartile for estrone (E₁) or ERα-mediated bioactivity had increased breast cancer risk. After additional adjustment for ERβ bioactivity, free estradiol, and E₁levels, serum ERα-mediated bioactivity remained associated with increased breast cancer risk. Compared with those in the lowest quartile, women in the highest quartile for ERα-mediated bioactivity had an odds ratio of 2.39 (95% CI=1.17–4.88;Pfor trend=0.016). Conversely, the positive association between E₁and cancer risk became null after adjustment for ERα-mediated bioactivity, suggesting that the effect of E₁could be mediated through ERα. Factor(s) contributing to increased ERα-mediated estrogenic bioactivity in serum and its role as a predictor for breast cancer risk need to be validated in future studies.