Objective: This study evaluated in obese rats the effect of exercise training on eNOS expressed in perivascular adipose tissue (PVAT) and its consequences on vascular function. Methods: Wistar rats were divided in 3 groups: control (standard diet), obese (high fat/high sucrose diet, HFS for 15 weeks), and exercised obese (HFS diet and exercise from week 6 to week 15, HFS-Ex) rats. The eNOS-adiponectin pathway and reactive oxygen species (ROS) were evaluated. Vascular reactivity was assessed on isolated aortic rings with or without PVAT and/or endothelium and exposed or not to the conditioned media of PVAT. Results: Obesity reduced eNOS level and phosphorylation on its activation site in the PVAT and had no impact on the vascular wall. Exercise training was able to increase eNOS and P-eNOS both in the vascular wall and in the PVAT. Interestingly, this was associated with increased level of adiponectin in the PVAT and to lower ROS in the vascular wall. Finally, PVAT of HFS-Ex aorta has eNOS-dependent anticontractile effects on endothelium denuded aortic rings and has beneficial effects on the endothelium-dependent vasorelaxation to ACh.