Understanding endothelial dysfunction is at the heart of current concerns due to its involvement in many cardiovascular diseases such as heart failure (HF). The β3-adrenergic receptor (β3-AR) could be at the origin of this endothelial dysfunction. Indeed, it is expressed at the level of the endothelial cell and induces vasorelaxation by the release of nitric oxide (NO), a central actor in endothelial dysfunction. Our objective was therefore to study the overexpression of the receptor in endothelial cells to understand its involvement in endothelial dysfunction. We used the Tgβ3 rat at 45 weeks of age, which overexpresses the human β3-AR gene in the endothelium. Endothelium-dependent aortic relaxation versus isoproterenol was measured with or without L-VNIO an inhibitor of nNOS at 10 μM. The protein expression of NOS was evaluated by Western-Blot and the production of NO and O2- was evaluated by EPR on aorta. Vascular reactivity was significantly reduced in response to isoproterenol in Tgβ3 aorta (−10% P < 0.05 vs. WT) indicating an alteration of endothelial function without alteration in systemic arterial pressure. With L-VNIO vasorelaxation was significantly reduce in Tgβ3 rat indicating a major implication of nNOS in vascular reactivity. nNOS expression was increased in aorta (+ 56% P < 0.05 vs. WT). Vascular production of NO was increased in Tgβ3 rats (+ 53% P < 0.05 vs. WT), possibly link to nNOS increase. This production was associated with an elevation of O2- and ONOO- in Tgβ3 rats (+ 340%, respectively P < 0.05 vs. WT). The increase in β3-AR expression induces endothelial dysfunction with a key role of eNOS and nNOS. Further studies will be conducted to understand the causal links between endothelial dysfunction and the alteration of the cardiac function.