Objective: To evaluate the association between estrogen (E) exposure and deficiency and cardiovascular disease (CVD) risk among women with primary ovarian insufficiency (POI). Design: Cross-sectional study conducted between 1996 and 2016. Setting: Tertiary referral centers. Patient(s): A total of 385 women with POI, defined by amenorrhea and FSH levels ≥40 IU/L before 40 years of age, were recruited. Intervention(s): None. Main Outcome Measure(s): Women underwent a standardized intake questionnaire including data on menstrual cyclicity. Lifetime E exposure and E-free period were assessed. Serum was analyzed for endocrine and CVD profiles. The Framingham 30-year risk of CVD was calculated. Result(s): Lifetime E exposure (mean ± SD) was 19.3 ± 7.0 years, E-free period was 3.1 ± 4.1 years, and age at screening was 34.8 ± 7.4 years. In multivariate models E-free interval associated positively with estimated risk of hard and general CVD events (β 0.18 [95% confidence interval 0.08, 0.29]; 0.20 [0.05, 0.35], respectively), and lifetime E exposure associated negatively with estimated risk of hard and general CVD events (−0.15 [−0.24, −0.05]; −0.16 [−0.29, −0.03], respectively), as well as low density lipoprotein cholesterol (−0.03 [−0.06, 0.00]) and non–high density lipoprotein cholesterol (−0.04 [−0.07, 0.00]). Conclusion(s): Prolonged E deprivation is associated with an increased estimated risk of CVD, whereas prolonged E exposure is associated with a reduced estimated risk. These results support the policy of early and continued use of E replacement therapy in women with POI. Clinical Trial Registration Number: NCT0230904.