Tumor necrosis factor (TNF) is one of the inflammatory cytokines which has an important role in inflammation and migration of other inflammatory cells to the atherosclerotic plaques. OX40 ligand (OX40L) is a member of the TNF super family receptor protein. OX40 and OX40L are co-stimulators for T-cells and can increase inflammatory response in atherosclerotic plaques. The aim of this study was to determine the association of rs3850641 polymorphism in OX40L gene with premature myocardial infarction (MI) in Iranian population. This case control study was done on 100 patients with premature MI and a similar number of sex, age and some other cardiovascular risk factor matched healthy people. The OX40L rs3850641 polymorphism was genotyped, using PCR-RFLP method. A-allele frequency of rs3850641 SNP was lower non-significantly in Premature MI, compared to healthy subjects (57.5% vs. 58.8%). The analysis of rs3850641 (A/G) polymorphism showed an odds ratio of 0.980 (95% CI: 0.473-2.030; P= 0.957) for the GG genotype and 1.127 (95% CI: 0.635-1.999; P= 0.00) for the AG genotype, compared to the AA genotype. The results of this study indicate that the rs3850641 SNP of OX40L gene is not associated with premature MI in the Iranian population.