Glibenclamide inhibits sulfonylurea receptor (SUR), which regulates several ion channels including SUR1-transient receptor potential melastatin 4 (SUR1-TRPM4) channel and ATP-sensitive potassium (K_ATP) channel. Stroke upregulates SURl-TRPM4 channel, which causes a rapid edema formation and brain swelling. Glibenclamide may antagonize the formation of cerebral edema during stroke. Preclinical studies showed that glibenclamide inhibits K_ATP channel-induced vasodilation without altering the basal vascular tone. The in vivo human cerebrovascular effects of glibenclamide have not previously been investigated. In a randomized, double-blind, placebo-controlled, three-way cross-over study, we used advanced 3 T MRI methods to investigate the effects of glibenclamide and K_ATP channel opener levcromakalim on mean global cerebral blood flow (CBF) and intra- and extracranial artery circumferences in 15 healthy volunteers. Glibenclamide administration did not alter the mean global CBF and the basal vascular tone. Following levcromakalim infusion, we observed a 14% increase of the mean global CBF and an 8% increase of middle cerebral artery (MCA) circumference, and glibenclamide did not attenuate levcromakalim-induced vascular changes. Collectively, the findings demonstrate the vital role of K_ATP channels in cerebrovascular hemodynamic and indicate that glibenclamide does not inhibit the protective effects of K_ATP channel activation during hypoxia and ischemia-induced brain injury.