Breast cancer (BC) is one of the most common causes of death among women, and second in Iran. The objectives of this study were to determine the frequency of methyltetrahydrofolate-homocysteine methyltransferase (MTR) 2756 gene polymorphism in patients with breast cancer. For the first time, we evaluated these polymorphisms and effects on the breast cancer risk association in an Iranian sporadic population-based case-control study of 282 breast cancer cases and 310 controls using a PCR-RFLP-based assay. Analyses of affected and controls show that homozygote genotype MTR 2756 AA has the highest frequency in both groups (33.3 in patients). Genotype MTR 2756 GG was the highest risk factor in our population [AG/GG odds ratio, 0.329 (95% CI: 0.146-0.741) p = 0.006, AA/AG, OR, 2.316, 95% CI: 1.509-3.555, p = 0.001, AA/GG odds ratio, 0.761 (95% CI: 0.363-1.595) p = 0.297]. There was a significant association of breast cancer risk with MTR 2756 GG and AA polymorphism.