BackgroundIt is well established that estrogens and other hormonal factors influence breast cancer susceptibility. We hypothesized that a woman’s total lifetime estrogen exposure accumulates changes in DNA methylation, detectable in the blood, which could be used in risk assessment for breast cancer.MethodsAn estimated lifetime estrogen exposure (ELEE) model was defined using epidemiological data from EPIC-Italy (n = 31,864). An epigenome-wide association study (EWAS) of ELEE was performed using existing Illumina HumanMethylation450K Beadchip (HM450K) methylation data obtained from EPIC-Italy blood DNA samples (n = 216). A methylation index (MI) of ELEE based on 31 CpG sites was developed using HM450K data from EPIC-Italy and the Generations Study and evaluated for association with breast cancer risk in an independent dataset from the Generations Study (n = 440 incident breast cancer cases matched to 440 healthy controls) using targeted bisulfite sequencing. Lastly, a meta-analysis was conducted including three additional cohorts, consisting of 1187 case-control pairs.ResultsWe observed an estimated 5% increase in breast cancer risk per 1-year longer ELEE (OR = 1.05, 95% CI 1.04–1.07, P = 3 × 10−12) in EPIC-Italy. The EWAS identified 694 CpG sites associated with ELEE (FDR Q < 0.05). We report a DNA methylation index (MI) associated with breast cancer risk that is validated in the Generations Study targeted bisulfite sequencing data (ORQ4_vs_Q1 = 1.77, 95% CI 1.07–2.93, P = 0.027) and in the meta-analysis (ORQ4_vs_Q1 = 1.43, 95% CI 1.05–2.00, P = 0.024); however, the correlation between the MI and ELEE was not validated across study cohorts.ConclusionWe have identified a blood DNA methylation signature associated with breast cancer risk in this study. Further investigation is required to confirm the interaction between estrogen exposure and DNA methylation in the blood.