Gene-targeted deletion of the immediate early responsive gene X-1 (IEX-1) results in a significant increase in systemic arterial blood pressure, but the underlying mechanism is not understood. Studies of arterial reactivity in isolated aortas revealed normal endothelium-dependent and -independent vasorelaxation and vasoconstriction but reduced cAMP-dependent vasorelaxation in the absence of IEX-1. This defect in cAMP signaling was also evident in endothelium-denuded aortic rings, consistent with the enhancement of mitochondrial O2·- production only in IEX-1-deficient vascular smooth muscle cells, not in endothelial cells. Excessive production of reactive oxygen species at mitochondria augmented the expression of Gα(i2), suppressing cAMP production in vascular smooth muscle cells. The role of mitochondrial reactive oxygen species in the upregulation of Gα(i2) leading to the development of hypertension was supported by the ability of antioxidant or pertussis toxin to restore the cAMP-dependent vasorelaxation to a normal level and reverse established hypertension in IEX-1 homozygous knockout mice. Our results suggest that hypertension in IEX-1 knockout mice may arise primarily from impaired cAMP signaling induced by overproduction of mitochondrial reactive oxygen species in vascular smooth muscle cells and demonstrate a causal relationship between mitochondrial dysfunction and cAMP-dependent vasorelaxation.