Aim: To assess the epistatic relationships of nitric oxide (NO) biosynthesis pathway genes in susceptibility to coronary heart disease (CHD). Methods: A total of 2142 subjects enrolled in two case-control studies was genotyped for 7 single-nucleotide polymorphisms (SNP) within NO biosynthesis pathway genes using TaqMan assays. The association analyses were performed at both SNP and haplotype levels. Two-way SNP-SNP interactions and high-order interactions were tested using multiple unconditional logistic regression analyses and generalized multifactor dimensionality reduction (GMDR) analyses, respectively. Results: Two alleles (rs1049255*C and rs841*A) were identified that were significantly associated with increased risk of CHD after adjusting for all confounders (OR=1.21, 95% CI: 1.06-1.39, combined P=0.001, P(corr)= 0.007 and OR=1.30, 95% CI 1.12-1.50, combined P<0.001, P(corr)< 0.001, respectively). Significant two-way SNP-SNP interactions were found between SNP rs2297518 and these two significant polymorphisms, affecting the risk of CHD (P<0.001 for both). No significant high-order interactions were identified. Conclusion: The results suggested that two-way SNP-SNP interactions of polymorphisms within NO biosynthesis pathway genes contribute to CHD risk.