Background: The true association between breast cancer and vitamin D is currently under investigation. We compared serum 25-hydroxy-vitamin D levels in women with benign and malignant breast masses and controls. Materials and Methods: Levels of vitamin D were measured by electrochemiluminescense. Serum levels >35 ng/ml, 25-35 ng/ml, 12.5-25 ng/ml and <12.5 ng/ml were considered as normal, mild, moderate and severe vitamin D deficiency, respectively. Results: Overall, 364 women were included in the control, 172 in the benign and 136 in the malignant groups. The median serum vitamin D level was significantly lower in breast cancers than controls. Levels were also lower in malignant than benign cases and in benign cases than controls although statistically non-significant. Conclusions: Multinomial logistic regression analysis showed that severe vitamin D deficiency causes a three-fold increase in the risk of breast cancer while this was not the case for moderate and mild deficiency.