Although, they are implicated on their own as risk factors for cardiovascular disease, the potential link between nitric oxide (NO) deficiency, angiotensin II (ANG II) and vascular stiffening has not been tested before. We evaluated the role of chronic ANG II treatment and NO deficiency, alone and in combination on aortic stiffness in mice and tested parameters contributing to increases in active or passive components of vascular stiffness, including blood pressure, vascular smooth muscle contractility, and extracellular matrix components. Untreated (control) mice and mice treated with a nitric oxide synthase inhibitor (L-NAME, 0.5 g/L) were implanted with osmotic minipumps delivering ANG II (500 ng/kg/min) for 28 days. Aortic stiffness was then measured in vivo by pulse wave velocity (PWV) and ex vivo by load-strain analysis to obtain values of maximal passive stiffness (MPS). Blood pressure and aortic contractility ex vivo were measured. ANG II treatment or NOS inhibition with L-NAME did not independently increase vascular stiffness, however the combined treatments worked synergistically to increase PWV and MPS. The combined treatments of ANG II + L-NAME also significantly increased aortic wall collagen content while decreasing elastin. These novel results suggest that NO deficiency and ANG II act synergistically to increase aortic stiffness in mice predominantly via changes in aortic wall collagen/elastin ratio.