To investigate the possible association between eNOS 4a/b polymorphism and the risk of developing type 2 diabetes mellitus (T2DM) and diabetic nephropathy (DN) 173 T2DM patients with and without DN and 101 healthy subjects with ethnic background of Kurds were examined for the frequency of eNOS variants using PCR-RFLP method. The frequency of eNOS 4a/b genotypes between T2DM and controls was not significantly difference. Studying eNOS 4a/b variants alone indicated that the presence of eNOS 4a allele was not associated with the risk of developing DN. However, considering both polymorphisms of eNOS 4a/b and G894T indicated that the risk of macroalbuminuria significantly increased in the presence of either eNOS 4a or 894T allele by 2.45 times (p=0.014) and 3.7-fold (p=0.016), respectively. However, the concomitant presence of both alleles was not associated with the risk of macroalbuminuria. In microalbuminuric patients, in the presence of each allele, the risk of microalbuminuria increased 2.2 times (p=0.028) and 2.72-fold (p=0.057) for eNOS 4a and 894T alleles, respectively. However, the combined presence of both eNOS 894T and 4a alleles was not associated with the risk of microalbuminuria. The present study indicates the absence of association between eNOS 4a/b variants and the risk of developing T2DM and DN. Also, we demonstrated that eNOS 4a or 894T allele alone increased the risk of developing DN but this effect was modified by the concomitant presence of both alleles.