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Introduction: Multiple Sclerosis (MS) is a disease of central nervous system that mainly causes lesions or plaques in the spinal cord and brain. The purpose of this study was to analyze the relation between c.-813C>T (rs2070744) and c.894G>T (rs1799983) polymorphisms of NOS3 gene and MS in Iranian patients. Methods: A total of 78 patients with MS and 80 healthy controls were screened for NOS3 (rs2070744 and rs1799983) Single Nucleotide Polymorphisms (SNPs) by tetra-primer multiplex ARMS-PCR and PCR-RFLP. Results: Genotype frequencies of the c.-813C>T polymorphism in patients compared to controls were as follows: 53.8% to 80.0% for TT genotype, 41.0% to 18.8% for TC genotype, and 5.1% versus 1.2% for CC genotype (P=0.001). The frequencies of GG genotype was 57.7% and 78.8% and for GT genotype of c.894G>T polymorphism in patients compared to control subjects was 42.3% and 21.2%, respectively (P=0.004). Conclusion: Our results indicate that the studied NOS3 polymorphisms may be associated with MS in Iranian patients.