ATP-sensitive K⁺ (K_ATP) channels mediate hypoxia-induced cerebral vasodilatation and hyperperfusion in animals. We tested whether K_ATP channels blockade affects the increase in cerebral blood flow (CBF) and the maintenance of oxygen delivery (CDO₂) during hypoxia in humans. Nine healthy men were exposed to 5-min trials of normoxia and isocapnic hypoxia (IHX, 10% O₂) before (BGB) and 3 h after glibenclamide ingestion (AGB). Mean arterial pressure (MAP), arterial saturation ( S_aO2), partial pressure of oxygen ( P_aO2) and carbon dioxide ( P_aCO2), internal carotid artery blood flow (ICABF), vertebral artery blood flow (VABF), total (t)CBF (Doppler ultrasound) and CDO₂ were quantified during the trials. IHX provoked similar reductions in S_aO2 and P_aO2, while MAP was not affected by oxygen desaturation or K_ATP blockade. A smaller increase in ICABF (ΔBGB: 36 ± 23 vs. ΔAGB 11 ± 18%, p = 0.019) but not in VABF (∆BGB 26 ± 21 vs. ∆AGB 27 ± 27%, p = 0.893) was observed during the hypoxic trial under KATP channels blockade. Thus, IHX-induced increases in tCBF (∆BGB 32 ± 19 vs. ∆AGB 14 ± 13%, p = 0.012) and CDO₂ relative changes (∆BGB 7 ± 13 vs. ∆AGB -6 ± 14%, p = 0.048) were attenuated during the AGB hypoxic trial. In a separate protocol, 6 healthy men (5 from protocol 1) underwent a 5-min exposure to normoxia and IHX before and 3 h after placebo (5 mg of cornstarch) ingestion. IHX reduced S_aO2 and P_aO2, but placebo did not affect the ICABF, VABF, tCBF, or CDO₂ responses. Therefore, in humans, K_ATP channels activation modulates vascular tone in the anterior rather than the posterior circulation of the brain, contributing to tCBF and CDO2 responses to hypoxia.