Cigarette smoke exposure (CSE) is one of the major risk factors of cardiovascular disease. Although tobacco smoking is associated with the onset of atherosclerosis, coronary disease, and ischemic heart disease, the precise mechanism by which these deleterious effects occur is not fully elucidated. In the current study, we provide evidence that CSE greatly impairs vascular endothelial function and induces cardiovascular changes in C57BL/6 male mice. Animals were exposed for a period of up to 48 weeks to the whole body mainstream and the side Stream cigarette smoke generated from 3R4F reference research cigarettes using the TE-10 cigarette smoking machine (Teague Enterprises, California). HPLC analysis revealed a significant decrease in tetrahydrobiopterin (BH4) level following CSE, along with a decrease in the expression levels of endothelial nitric oxide synthase (eNOS) and its Ser¹¹¹⁷phosphorylated (activated) form in the aorta of smoke-exposed mice. Consistently, immunohistochemical analysis showed a clear decrease in eNOS expression in the endothelium of aorta from smoke-exposed mice compared to controls. Smoke-exposed mice also showed overexpression of NADPH oxidase, giving rise to reactive oxygen species (ROS) production and inducing BH4 depletion. CSE impaired acetylcholine-induced vascular relaxation and induced persistent hypertension and cardiac hypertrophy in smoke-exposed mice. Our data indicate that CSE decreases BH4 bioavailability, resulting in eNOS uncoupling, which triggers ROS generation and decreased NO production leading to vascular endothelial dysfunction, hypertension and cardiac hypertrophy. Overall, our study provides important insights toward understanding how smoking contributes to the genesis of cardiovascular disease.