Abstract Body: Clinical interest in circulating adipocyte-derived extracellular vesicles (Ad-EVs) has intensified due to their potential as a circulating biomarker and mediator of cardiometabolic health and disease. We have shown that circulating Ad-EVs are elevated with obesity and associated with endothelial dysfunction; however, the underlying mechanisms are unknown. The experimental aim of this study was to determine the effect of Ad-EVs isolated from adults with obesity on endothelial cell inflammation, oxidative stress, nitric oxide (NO) and endothelin (ET)-1 production. Thirty-two sedentary, otherwise healthy midlife adults (43-66 years) were studied: 16 normal weight (9M/7F; age: 56±2 yr; BMI: 21.7±0.5 kg/m²; Ad-EVs: 115±9 Ad-EV/µL) and 16 obese (9M/7F; 56±2 yr; 32.0±0.6 kg/m²; 436±51Ad-EV/µL). Ad-EVs (perilipin A⁺vesicles) were identified, enumerated, and isolated from plasma by flow cytometry. Human coronary artery endothelial cells (HCAECs) were cultured and treated with Ad-EVs from either normal weight adults or adults with obesity. Although intracellular expression of the inflammation transcription factor NF-kB p65 (110.6±7.8 vs 112.0±7.0 AU) was not significantly affected, Ad-EVs from adults with obesity induced ~25% higher (P<0.05) expression of p-NF-kB p65 (40.7±2.3 vs 30.2±2.2 AU). Intracellular reactive oxygen species (ROS) production was significantly higher (~55%) in HCAECs treated with Ad-EVs from obese (164±11%) vs normal weight (111±10%) adults. Concordantly, antioxidant proteins SOD-1 (197.8±10.4 vs 254.4±12.7 AU) and catalase (40.7±5.2 vs 82.7±4.7 AU) were ~40% lower (P<0.01) in cells treated with obesity-related Ad-EVs. Expression of p-eNOS (Ser1177) was lower (27.5±1.9 vs 43.1±2.3 AU; P=0.001) and p-eNOS (Thr495) higher (15.2±0.9 vs 8.4±0.6 AU; P<0.001); in addition, expression of Big ET-1 (84.3±4.4 vs 75.7±2.7 AU) and endothelin converting enzyme (417.8±30.9 vs 344.1±8.0 AU) were also higher in cells treated with Ad-EVs from obese vs normal weight adults. As a result, NO production was lower (5.8±0.1 vs 6.9±0.2 μmol/L; P=0.02) and ET-1 production higher (337.8±25.0 vs 257.6±30.0 pg/mL; P=0.04) in cells treated with Ad-EVs from obese adults. Increased endothelial inflammation, oxidative stress and ET-1 production coupled with impaired NO production renders the vasculature prone to atherogenesis. Circulating Ad-EVs represent novel mediators of obesity-related endothelial dysfunction and, in turn, increased CVD risk.