Objective S-adenosylmethionine (SAM) is a key intermediate in the metabolism of sulfur amino acids, and is a major methyl donor in the cell. Although low plasma SAM level was suggested to be associated with atherosclerosis, the effect of SAM administration on atherosclerosis is not established. This study was undertaken to investigate the possible preventive effect of SAM on atherosclerosis and its molecular mechanism. Methods Eight-week-old male Sprague-Dawley (SD) rats were given normal rat chow or a high fat diet (HFD) with or without 30 mg/kg/day of SAM. In addition, eight-week-old male ApoE^-/- mice on the C57BL/6 background received an intraperitoneal injection of 4-mg/kg of streptozotocin for 5 consecutive days. Both STZ-administered and control ApoE^-/- mice were maintained on regular chow until 4 weeks after starting the STZ administration, when they were switched to HFD for 16 weeks with or without 30 mg/kg/day SAM. Results SAM treatment prevented endothelial dysfunction in HFD-fed rats, and prevented atherosclerosis in diabetic Apo E-/- mice given HFD. In cultured human aortic endothelial cells, SAM prevented linoleic acid (LA)-induced cell apoptosis and endoplasmic reticulum (ER) stress. SAM increased heme oxygenase-1 (HO-1) expression in an NF-E2-related factor 2-dependent manner. siRNA against HO-1 reversed SAM’s effects on cell apoptosis. Conclusion These data demonstrate that SAM prevents vascular dysfunction and atherosclerosis in HFD-fed animals. In cultured endothelial cells, SAM induced HO-1, ameliorated ER stress and prevented cell apoptosis. SAM treatment may be a new therapeutic strategy for atherosclerosis.