Background: Although some observational studies have associated higher calcium intake and especially higher vitamin D intake and 25-hydroxyvitamin D levels with lower breast cancer risk, no randomized trial has evaluated these relationships. Methods: Postmenopausal women (N = 36 282) who were enrolled in a Women's Health Initiative clinical trial were randomly assigned to 1000 mg of elemental calcium with 400 IU of vitamin D3 daily or placebo for a mean of 7.0 years to determine the effects of supplement use on incidence of hip fracture. Mammograms and breast exams were serially conducted. Invasive breast cancer was a secondary outcome. Baseline serum 25-hydroxyvitamin D levels were assessed in a nested case-control study of 1067 case patients and 1067 control subjects. A Cox proportional hazards model was used to estimate the risk of breast cancer associated with random assignment to calcium with vitamin D 3. Associations between 25-hydroxyvitamin D serum levels and total vitamin D intake, body mass index (BMI), recreational physical activity, and breast cancer risks were evaluated using logistic regression models. Statistical tests were two-sided. Results: Invasive breast cancer incidence was similar in the two groups (528 supplement vs 546 placebo; hazard ratio = 0.96; 95% confidence interval = 0.85 to 1.09). In the nested case-control study, no effect of supplement group assignment on breast cancer risk was seen. Baseline 25-hydroxyvitamin D levels were modestly correlated with total vitamin D intake (diet and supplements) (r = 0.19, P <. 001) and were higher among women with lower BMI and higher recreational physical activity (both P <. 001). Baseline 25-hydroxyvitamin D levels were not associated with breast cancer risk in analyses that were adjusted for BMI and physical activity (Ptrend =. 20). Conclusions: Calcium and vitamin D supplementation did not reduce invasive breast cancer incidence in postmenopausal women. In addition, 25-hydroxyvitamin D levels were not associated with subsequent breast cancer risk. These findings do not support a relationship between total vitamin D intake and 25-hydroxyvitamin D levels with breast cancer risk. © The Author 2008. Published by Oxford University Press. All rights reserved.