High altitude trekking provokes reductions in flow-mediated dilation (FMD) and compensatory adjustments in cerebral blood flow (CBF) to maintain cerebral oxygen delivery. However, the impact of acute restoration of oxygen on these responses is unclear. We investigated whether oxygen supplementation (O2), to acutely relieve hypoxemia, would reverse alterations in FMD and CBF. Healthy, male participants (aged 21-42 years) were examined at 1400m (baseline), prior to and during O2 (after a 15-minute wash-in; target oxyhaemoglobin saturation of 99%) at 3440m (day 4 at high altitude; n=7), 4371m (day 7; n=7), and 5050m (day 10; n=12). Duplex ultrasound was performed to assess blood flow and FMD in the brachial artery and derive CBF from internal carotid artery and vertebral artery blood flows. Oxygen delivery was calculated as estimated O2 content × blood flow. Brachial artery blood flow was lower at all locations compared to baseline. FMD was reduced and CBF augmented only at 5050m compared to baseline (P=0.002 and 0.005). At 3440m, O2 decreased brachial artery diameter by 5±5% (P=0.04), reduced blood flow by 44±15% (P<0.001), O2 delivery by 37±16% (P<0.001) and improved FMD (P=0.04); however, the increased FMD was explained by the reductions in baseline diameter. No brachial artery parameters were affected by O2 at 4371m. A reduction in brachial artery blood flow (-17±22%; P=0.03), but not oxygen delivery or diameter, occurred with O2 at 5050m. There was a trend towards increased FMD with O2 at 5050m (3.4±2.2% to 4.8±1.7%; P=0.07). CBF was unaffected by O2 at 3440m or 4371m. At 5050m, internal carotid artery blood flow and CBF decreased by 9±13% (P=0.04) and 13±11% (P=0.009), respectively, with O2; nevertheless, estimated cerebral oxygen delivery was elevated by 14±14% during O2 (P=0.02). Collectively, these findings suggest that at initial modest altitude O2 abrogates upper limb hypoxic vasodilation along the arterial tree (i.e. conduit and resistance arteries) during early ascent. With prolonged, more severe high-altitude exposure, O2 alleviates hypoxic vasodilation in the upper limb and cerebral circulations whilst augmenting FMD, conceivably by reducing sympathetic vasomotor tone.