Objective(s): One of the most important problems of taking nitroglycerin is the nitrate tolerance phenomenon and endothelial dysfunction. Oxidative stress is a high-emphasized one of tolerance mechanisms. The possible effect of crocin, one of the anti-oxidant ingredients of saffron, on the nitrate tolerance model was investigated.Materials and Methods: In the present study, lipid peroxidation and the level of activated and deactivated forms of eNOS were measured. Animals were administered subcutaneously with 25 mg/kg of nitroglycerin, twice a day for 3 days to induce nitrate tolerance model. For evaluation of crocin effects, 20, 40 and 80 mg/kg/day of this compound were injected intraperitoneally in concomitant with nitroglycerin. In the isolated aorta test, after preparation of aorta rings, different concentrations of acetylcholine, sodium nitroprusside and nitroglycerin were added to the organ bath after inducing contraction by phenylephrine and the responsiveness of tissues was recorded.Results: Findings showed that nitroglycerin administration caused a remarkable overproduction of malondialdehyde (MDA) in the cells and crocin treatment significantly decreased the MDA level. In the nitrate tolerance group, the level of activated eNOS decreased and the level of deactivated eNOS increased. Crocin partly alleviated these changes: however, its effects were not remarkable. Nitroglycerin injection for 3 days developed tolerance to nitroglycerin and cross-tolerance to acetylcholine (endothelial dysfunction) and sodium nitroprusside. Crocin failed to influence significantly on the nitrate tolerance.Conclusion: Crocin effectiveness is possibly time-dependent; therefore, increasing the duration of treatment with crocin may lead to a significant prevention of nitrate tolerance and endothelial dysfunction.