Aim: To investigate the association of polymorphisms in four critical genes implicated in the NO-forming pathway with ischemic stroke (IS) in a Chinese Han population. Methods: DNA samples of 558 IS patients and 557 healthy controls from Chinese Han population were genotyped using the Taqman (TM) 7900HT Sequence Detection System. Six SNPs (rs841, rs1049255, rs2297518, rs1799983, rs2020744, rs4673) of the 4 related genes (eNOS, iNOS, GCH1, and CYBA) in the NO forming pathway were analyzed using the SPSS 13.0 software package for Windows. Results: One SNP located in the intron of GCH1 (rs841) was associated with IS independent of the traditional cardiovascular risk factors in co-dominant and dominant models (P=0.003, q=0.027; P=0.00006, q=0.0108; respectively). Moreover, the combination of rs1049255 CC+CT and rs841 GA+AA genotypes was associated with significantly higher risk for IS after adjustments (OR=1.73, 95% CI: 1.27-2.35, P<0.0001, q<0.0001). Conclusion: The data suggest that genetic variants within the NO-forming pathway alter susceptibility to IS in Chinese Han population. Replication of the present results in other independent cohorts is warranted.