• To investigate the association of the T-786C, G894T and variable number of tandem repeats (VNTRs) in intron 4 (a/b) polymorphisms of the eNOS gene in Iranian subjects with vasculogenic erectile dysfunction (ED). • A total of 322 consecutive patients with vasculogenic ED were recruited. Patients with concomitant risk factors for ED were excluded. • Patients with ED were identified based on history-taking, detailed physical examination, serum biochemistry, sex hormone measurements, application of the International Index of Erectile Function (IIEF) questionnaire, and penile duplex Doppler ultrasonography after intracavernosal injection of 20 µg prostaglandin E(1) . The control group comprised 318 age-matched healthy male volunteers. • Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism and the T-786C, G894T and VNTR intron 4 polymorphisms of the eNOS gene were determined. • After multivariate regression analysis, significant differences were seen in the frequencies of genotypes and alleles of the two T-786C and G894T polymorphisms when patients with ED and normal controls were compared. • In a multiple logistic regression analysis, the odds ratio (OR) of increased ED was strongly associated with the -786C allele [adjusted OR = 3.12, 95% confidence interval (CI) = 2.28-4.25; P= 0.001] and the 894T allele (adjusted OR = 3.87, 95% CI = 2.53-4.87; P= 0.001). • The data showed a higher prevalence of the T-786C CC genotype (adjusted OR = 2.72, 95% CI = 1.88-3.65; P= 0.006), and the G894T GT (adjusted OR = 1.72, 95% CI 1.24-2.83; P= 0.037) and G894T TT genotypes (adjusted OR = 3.42, 95% CI 2.42-4.26; P= 0.001) in patients with ED than in the controls. • The findings of the present study suggest that the eNOS T-786C and G894T polymorphisms are strong predictors of the predisposition to ED in addition to traditional risk factors, signifying a genetic influence for this multifactorial disease. • Further studies in different ethnic populations are needed to better elucidate the role of eNOS gene polymorphism in the pathogenesis of ED.