Recent studies have demonstrated that making the sorts of oxygen reactive, such as nitric oxide, can cause oxidative lipid damage, protein damage, and damage to the DNA of cells. Sperm DNA damage effect on the reduction of sperm mobility and damage of acrosome membrane lead to the inability of sperm to fertilize the oocyte. Increasing expression of endothelial nitric oxide synthase (eNOS) gene is seen in various diseases such as cardiovascular and infertility diseases. This study aimed to assess the association between eNOS gene single nucleotide polymorphism/SNP (rs2070744, T786C) and risk of male infertility and the quality of sperm parameters in an Iranian population. In this case-control study, 100 infertile men were enrolled as a patient group. Control groups consisted of 100 fertile men. T786C genotypes were determined using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Results showed that T786C SNP, contained frequent genotype TC (p = 0.000; OR = 0.000; 95% CI = 0.000-0.015), TC + CC genotypes (p = 0.000; OR = 0.000; 95% CI = 0.000-0.015), and C allele (p = 0.000; OR = 0.00; 95% CI = 0.000-0.007), revealed a significant with male infertility. Based on the findings of this study suggested that although T786C SNP could not be applied as an appropriate genetic risk factor for male infertility, it probably may be considered a protective marker for other researchers. However, more comprehensive studies in different populations are required to confirm our data.