Background: Impaired function of endothelial nitric oxide synthase (eNOS) is involved in the pathologic processes of erectile dysfunction (ED), and three functional polymorphisms (G894T, T-786C, and a tandem repeat of 27 bp in intron 4) in the NOS3 gene, which encodes eNOS, are associated with the clinical characteristics of ED in several populations. Aim: To investigate the effect of these variations of NOS3 on ED phenotypes and the response to sildenafil in a Han Chinese population. Methods: This case-control study enrolled 112 patients with ED and 156 age-matched healthy men. Their medical history and laboratory data were collected. ED severity and response to sildenafil were assessed using the five-item International Index of Erectile Function (IIEF-5) score. Routine polymerase chain reaction and Sanger sequencing were used to genotype the three polymorphisms of NOS3. Outcomes: The frequencies of alleles, genotypes, and haplotypes of the loci in patients and controls; the IIEF-5 scores of patients carrying the risk and non-risk genotype; and the frequencies of risk and non-risk genotypes in patients with different ages at onset and responses to sildenafil were assessed. Results: The frequencies of drinkers and diabetic and hyperlipidemic patients in the ED group were higher than those in the age-matched control group (P<.05). The distributions of alleles (G894T, P<.005; T-786C, P<.015), genotypes (G894T, P<0.015; T-786C, P<.010), and haplotypes (G894T/T-786C, P<.015) of the NOS3 polymorphisms were significantly different between patients with ED and controls. An increased risk for earlier onset of ED was observed in the G894T risk genotype carriers (odds ratio = 3.572; P<.020). Patients with the risk genotype of T-786C exhibited lower IIEF-5 scores than patients with the non-risk genotype (8.2 +/- 4.5 vs 12.2 +/- 5.0; P<.015). The influence of the T-786C or G894T genotype on the response to sildenafil was not observed. Clinical Translation: The detectable effect of NOS3 functional polymorphisms on ED suggests their application potential as a molecular biomarker in predicting ED susceptibility and severity in the Han Chinese population. Strengths & Limitations: This study provides strong evidence that NOS3 functional variation is an independent risk factor for ED in the Han Chinese population, which should be confirmed in larger cohorts considering the limited number of subjects in this study. Conclusion: These results are the first to identify a clear association between NOS3 functional variation and ED susceptibility, age at onset, and severity in the Han Chinese population.