Background: High 25-hydroxyvitamin D [25(OH)D] serum concentrations have been found to be associated with reduced breast cancer risk. However, few studies have further investigated this relationship according to menopausal status, nor have they taken into account factors known to influence vitamin D status, such as dietary and serum calcium, parathyroid hormone, and estradiol serum levels. Methods: We designed a nested case-control study within the French E3N cohort. Cases were women diagnosed with incident breast cancer (n = 636). Controls (n = 1,272) were matched with cases on age, menopausal status at blood collection, age at menopause, and center and year of blood collection. Multivariate logistic regression models were established. Results: We found a decreased risk of breast cancer with increasing 25(OH) vitamin D3 serum concentrations (odds ratio, 0.73; 95% confidence interval, 0.55-0.96; P trend = 0.02) among women in the highest tertile. We also observed a significant inverse association restricted to women under 53 years of age at blood sampling [odds ratio (T3 versus T1), 0.60; 95% confidence interval, 0.37-0.98; P trend = 0.04]. In premenopausal women, the risk was also decreased, although not significantly. Conclusion: Our findings support a decreased risk of breast cancer associated with high 25(OH) vitamin D3 serum concentrations, especially in younger women, although we were unable to confirm a direct influence of age or menopausal status. Impact: Randomized intervention trials with vitamin D supplementation are required to confirm its benefits on breast cancer risk, but the maintenance of adequate vitamin D levels should be encouraged by public health policy.