In testis, eNOS is responsible for synthesis of nitric oxide (NO) which is an essential gas message regulator in spermatogenesis, suggesting that eNOS gene plays a role in normal spermatogenesis and the genetic variants of eNOS gene may be potential genetic risk factors of spermatogenesis impairment. In this study, the polymorphic distributions of three common polymorphism loci including T-786C, 4A4B and G894T in eNOS gene were investigated in 355 Chinese infertile patients with azoospermia or oligozoospermia and 246 healthy fertile men and a meta-analysis was carried in order to explore the possible relationship between the three loci of eNOS gene and male infertility with spermatogenesis impairment. As a result, allele -786C of T-786C (11.4% versus 6.5%, p = 0.004) and 4A of 4A4B (11.0% versus 6.3%, p = 0.005) as well as genotype TC of T-786C (22.8% versus 13.0%, p = 0.002) and AB of 4A4B (18% versus 11%, p = 0.015) were significantly associated with idiopathic male infertility. The haplotypes T-4A-G (7.4% versus 4.1%, p = 0.015) and C-4B-G (7.6% versus 4.4%, p = 0.028) could increase the susceptibility to male infertility, whereas haplotype T-4B-G (67.0% versus 75.2%, p = 0.002) might be a protective factor for male infertility. The results of meta-analysis revealed that the polymorphism of T-786C was associated with male infertility. These findings suggested that the variants of eNOS gene may modify the susceptibility to male infertility with impaired spermatogenesis.