The present study was designed to evaluate the possible in vivo protective effects of sesamin on hypertension and endothelial function in two-kidney, one-clip renovascular hypertensive rats fed with a high-fat, high-sucrose diet (2K1C rats on HFS diet). Sesamin was orally administered for 8 weeks in 2K1C rats on HFS diet. Then, the serum malondialdehyde level was determined. The protein expression of endothelial nitric oxide synthase (eNOS), nitrotyrosine and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit p47(phox) in aortas was detected by Western blotting. Vasorelaxation response to acetylcholine and nitroprusside, and functional assessment of nitric oxide (NO) bioactivity were also determined in aortic rings. Sesamin treatment reduced systolic blood pressure, improved vasodilatation induced by acetylcholine and enhanced NO bioactivity in the thoracic aortas. These changes were associated with increased eNOS, decreased malondialdehyde content, and reduced nitrotyrosine and p47(phox) protein expression. All these results suggest that chronic treatment with sesamin reduces hypertension and improves endothelial dysfunction through upregulation of eNOS expression and reduction of NO oxidative inactivation in 2K1C rats on HFS diet.