3,4^′,5-trimethoxy-trans-stilbene (TMS) is a methoxylated derivative of resveratrol. Previous studies showed the vaso-protective effects of resveratrol; nevertheless, research on this derivative is scarce. The current study aimed to explore whether TMS can alleviate endothelial dysfunction in diabetic and obese mice, along with the underlying mechanisms. Thoracic aortas isolated from male C57BL/6J mice and primary cultures of rat aortic endothelial cells were treated with high glucose with or without TMS. High glucose exposure impaired acetylcholine-induced endotheliumdependent relaxations, down-regulated NO bioavailability and the AMP-activated protein kinase (AMPK)/Sirtuin 1 (SIRT1)/endothelial nitric oxide synthase (eNOS) pathway, increased endoplasmic reticulum (ER) stress and oxidative stress, which were reversed by TMS treatment. Moreover, the protective effects of TMS were abolished by Compound C (AMPK inhibitor), and EX527 (SIRT1 inhibitor). The mice were fed with high-fat diet (60% kcal% fat) for 14 weeks to establish a diabetic and obese model, and were orally administered TMS (10 mg/kg/day) in the last 4 weeks. Chronic TMS treatment alleviated endothelial dysfunction via enhancing the AMPK/SIRT1/eNOS pathway and attenuated oxidative stress and ER stress in aortas of diet-induced obese mice. In summary, our study reveals the potent vaso-protective effect of TMS and its therapeutic potential against endothelial dysfunction in metabolic disorders. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.