There were some case-control studies, nested case-control studies, and cohort studies with controversial results on the association between serum 25-hydroxyvitamin D [25(OH)D] and breast cancer risk. Case-control studies are prone to selection bias, which limit the strength and quality of the evidence. To overcome the shortcoming of the case-control studies, the meta-analysis of prospective studies including nested case-control studies and cohort studies was conducted. PubMed, Embase, and Web of Science databases were searched, and the last retrieval date was March 24, 2013. For the highest versus the lowest level of serum 25(OH)D, the relative risks (RRs) and its 95% confidence intervals (CIs) from each study were used to estimate summary RR and its 95% CI. Subgroup analyses by geographic region, menopausal status, and adjusted status of RR were also performed, respectively. A dose-response association between serum 25(OH)D concentration and breast cancer risk was assessed. Fourteen articles with 9,110 breast cancer cases and 16,244 controls were included in the meta-analysis. Overall, serum 25(OH)D levels were inversely significantly associated with breast cancer risk (RR = 0.845, 95% CI = 0.750-0.951). Inversely statistically significant associations were observed in North American studies, postmenopausal women, and studies with adjusted and unadjusted RR, respectively. No statistically significant associations were observed in European studies and premenopausal women, respectively. Dose-response analysis showed that every 10 ng/mL increment in serum 25(OH)D concentration was associated with a significant 3.2% reduction in breast cancer risk. This meta-analysis provides evidence of a significantly inverse association between serum 25(OH)D concentration and breast cancer risk.