Diazoxide (DIAZ), an activator of mitochondrial ATP-sensitive potassium (mitoK_ATP) channels, is neuroprotective, but the mechanism of action is unclear. We tested if DIAZ preserves endothelium-dependent (hypercapnia) or -independent (iloprost, ILO) cerebrovascular dilator responses following ischemia/reperfusion (I/R) in newborn pigs, and whether the effect of DIAZ is sensitive to 5-hydroxydecanoate (5HD), an inhibitor of mitoK_ATP. Anesthetized, ventilated piglets (n=48) were equipped with closed cranial windows. The changes in diameter of pial arterioles were determined with intravital microscopy in response to graded hypercapnia (5-10% CO₂, 21% O₂, balance N₂; n=25) or ILO (0.1-1 µg/mL; n=18) before and 1 h after 10 min of global I/R. The experimental groups were pretreated with vehicle, NS-398, a selective cyclooxygenase-2 inhibitor (1 mg/kg), DIAZ (3 mg/kg), or 5HD(20 mg/kg)+DIAZ. The potential direct effects of DIAZ and 5HD on hypercapnic vasodilation were also tested in the absence of I/R (n=5). To confirm the direct effect of DIAZ on mitochondria, mitochondrial membrane potential in cultured piglet cerebrovascular endothelial cells was was monitored using MitoTracker Red. Hypercapnia resulted in dose-dependent pial arteriolar vasodilation, which was attenuated by ~70% after I/R both in the vehicle-treated and in the NS-398-treated animals. DIAZ and 5HD did not affect the CO₂ response. DIAZ significantly preserved the postischemic vasodilation to hypercapnia but not to ILO. DIAZ depolarized mitochondria in cultured piglet cerebrovascular endothelial cells, and 5HD completely abolished the protective effect of DIAZ, both indicating a role for mitoK_ATP. In summary, preservation of arteriolar dilator responsiveness by DIAZ may contribute to neuroprotection.