NBS1, also known as NBN, plays an important role in maintaining genomic stability. Interestingly, rs2735383 G > C, located in a microRNA binding site in the 3′-untranslated region (UTR) of NBS1, was shown to be associated with increased susceptibility to lung and colorectal cancer. However, the relation between rs2735383 and susceptibility to breast cancer is not yet clear. Therefore, we genotyped rs2735383 in 1,170 familial non-BRCA1/2 breast cancer cases and 1,077 controls using PCR-based restriction fragment length polymorphism (RFLP-PCR) analysis, but found no association between rs2735383CC and breast cancer risk (OR = 1.214, 95% CI = 0.936–1.574, P = 0.144). Because we could not exclude a small effect size due to a limited sample size, we further analyzed imputed rs2735383 genotypes (r2 > 0.999) of 47,640 breast cancer cases and 46,656 controls from the Breast Cancer Association Consortium (BCAC). However, rs2735383CC was not associated with overall breast cancer risk in European (OR = 1.014, 95% CI = 0.969–1.060, P = 0.556) nor in Asian women (OR = 0.998, 95% CI = 0.905–1.100, P = 0.961). Subgroup analyses by age, age at menarche, age at menopause, menopausal status, number of pregnancies, breast feeding, family history and receptor status also did not reveal a significant association. This study therefore does not support the involvement of the genotype at NBS1 rs2735383 in breast cancer susceptibility.