Background As breast epithelium is affected by vitamin D, it may have a direct effect on breast density and the risk of breast cancer. Our aim was to study the serum levels of vitamin D in patients with malignant and benign breast disease, and to study the association, if any, between vitamin D levels, mammographic breast density (MD) and molecular subtypes of breast cancer. Methods In this cross-sectional, observational study, we enrolled 162 consecutive adult women with benign and malignant breast masses subjected to mammography and core-needle biopsy. Serum levels of vitamin D were estimated and correlated with MD and with immunohistochemical subtyping of breast cancer. Results The mean vitamin D level in these 162 patients was 12.44 (5.88) ng/ml, with vitamin D deficiency seen in 98%. The mean (SD) vitamin D level in MD type 1 was 16.19 (4.62) ng/ml and it decreased to 7.54 (2.58) ng/ml in MD type 4. High MD was associated with significantly lower vitamin D levels. The mean vitamin D level in patients with benign breast disease (n=102) was 13.73 (5.68) ng/ml, while it was significantly lower in patients with breast cancer (n=60) at 10.26 (5.61) ng/ml. Among patients with breast cancer, the good prognosis luminal A molecular subtype had mean vitamin D level of 12.94 (6.16) ng/ml, whereas the poor prognosis triple-negative subtype had a significantly lower value of 7.68 (3.42) ng/ml. Conclusion Our study shows that vitamin D deficiency has a significant relationship with breast cancer (v. benign breast disease), high MD (showing increased breast cancer risk) and poor prognosis triple-negative breast cancer. Vitamin D deficiency could be an important, potentially modifiable, risk factor for the prevention of breast cancer in susceptible populations.