Abstract Earlier age of menopause and bilateral oophorectomy are associated with accelerated biologic aging based on epigenetic clocks. While these relationships suggest women with greater epigenetic age acceleration (AgeAccel) might be at a reduced risk of postmenopausal breast cancer, prior studies conflict with this theory. We hypothesized this paradox may be attributable to an interaction between epigenetic and reproductive aging on cancer risk. We evaluated this premise among 5,044 postmenopausal women in the Women’s Health Initiative (WHI) Observational Study and Clinical Trial with AgeAccel estimated in whole blood. Among a subset of 1,135 of these women, estradiol (E2) and sex hormone-binding globulin were assayed in baseline serum samples. For WHI participants with DNA methylation assays, we modeled the log odds of incident postmenopausal breast cancer during follow-up as a function of AgeAccel, adjusting for age at menopause, race/ethnicity, age at WHI screening, bilateral oophorectomy, nulliparity, alcohol consumption, smoking, body mass index, duration of postmenopausal hormone therapy use, exercise, clinical trial arm, and hysterectomy status at baseline. We repeated this analysis among the subset of participants with DNA methylation and E2 assays, and appraised the degree to which bioavailable E2 levels contributed to the observed association between AgeAccel and incident postmenopausal breast cancer. Finally, we evaluated whether bioavailable E2 levels modified the relationship between AgeAccel and cancer risk. Generalized estimating equations were used to model associations with AgeAccel, integrating repeated measures among a subset of participants and using inverse probability weights to account for sample selection probabilities. Based on our fully adjusted models, increased extrinsic AgeAccel was associated with decreased odds of incident postmenopausal invasive breast cancer during follow-up. This association was consistent among the subset of participants with E2 assays, and robust to adjustment for bioavailable E2 concentration. We found the inverse relationship between extrinsic AgeAccel and incident breast cancer was strongest among white non-Hispanic women with low levels of bioavailable E2. This study represents the largest investigation of the association between AgeAccel and postmenopausal breast cancer risk, and the first evaluation of how bioavailable E2 levels may influence this relationship. Our analyses inform our understanding of the relationship between the epigenetic and reproductive aging process, and the potential implications for postmenopausal breast cancer risk. Citation Format: Alexandra M. Binder, Lesley Tinker, Robert Wallace, JoAnn E. Manson, Lihong Qi, Parveen Bhatti, Eric A. Whitsel, Andrea Z. LaCroix, Steve Horvath. Association between epigenetic age acceleration and postmenopausal breast cancer risk in the Women’s Health Initiative [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS7-28.