Objective. Mitochondria affect cerebral vascular tone by activation of mitochondrial ATP-sensitive potassium channels (mitoK_ATP) and the generation of reactive oxygen species (ROS). Insulin resistance (IR) accompanying obesity causes mitochondrial dysfunction but the consequences on the cerebral circulation have not been fully identified. We evaluated the mitochondrial effects of diazoxide, a putative mitoK_ATP activator, on cerebral arteries of Zucker obese (ZO) rats with IR and lean (ZL) controls. Methods and Results. Diameter measurements showed diminished diazoxide-induced vasodilation in ZO compared to ZL rats. Maximal relaxation (%) was 38±3 in ZL versus 21±4 in ZO (p<0.05). Iberiotoxin, a calcium-activated potassium channel (K_Ca) inhibitor or MnTBAP, a superoxide dismutase (SOD) mimetic or endothelial denudation, diminshed vasodilation to diazoxide in all arteries implicating K_Ca, ROS and endothelial factors in vasodilation. Inhibition of nitric oxide synthase (NOS) in ZL, diminished diazoxide-induced vasodilation in intact arteries but vasodilation was unaffected in endothelium-denuded arteries. In contrast, NOS inhibition in ZO, enhanced vasodilation in endothelium-denuded arteries but intact arteries were unaffected suggesting that activity of endothelial NOS was abolished while factors derived from non-endothelial NOS promoted vasoconstriction. Fluorescence microscopy showed decreased mitochondrial depolarization, ROS production and NO generation in response to diazoxide in ZO arteries. Protein and mRNA measurements revealed increased expression of eNOS and SODs in ZO arteries. Conclusions. Cerebrovascular dilation to mitochondria-derived factors involves the integration of endothelial and smooth muscle mechanisms. Furthermore, mitochondria-mediated vasodilation was diminished in ZO rats due to impaired mitoK_ATP activation, diminished ROS generation, increased ROS scavenging and abnormal NOS activity.