Cigarette smoking is a risk factor for cardiovascular disease. The relationship between smoking and cardiovascular disease results from multiple mechanisms that interact to contribute to several pathological conditions, including vascular dysfunction. In chronic smokers, reduced endothelial-derived nitric oxide (NO) bioactivity and impairment of endothelium-dependent vasodilation have been shown to be associated with cardiovascular disease risk factors. Here, we tested the hypothesis that cytoglobin, a newly discovered globin that degrades NO, plays a role in the regulation of NO bioactivity in aortas of cigarette smoke exposed mice. Male C57BL/6 mice were exposed for a period of 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). Chronic exposure of C57BL/6 mice to cigarette smoke resulted in elevation of the blood pressure in a time dependent manner. Such an effect was accompanied with impairment of acetylcholine-induced vascular response. Electrochemical measurement of NO revealed that the decay of NO was faster in smoke-exposed mice compared to non-exposed controls. Interestingly, immunohistochemical analysis of cytoglobin in mouse aortas showed that cytoglobin expression was higher in the smoke-exposed mice compared to the controls. Moreover, these data were supported by immunoblotting analysis of cytoglobin in aortic tissue homogenate. In conclusion, the data suggest that chronic cigarette smoke exposure induces vascular dysfunction and faster rate of NO decay, in part, through the upregulation of cytoglobin. Our study provides an important insight toward understanding how smoking contributes to the genesis of cardiovascular disease.