Human vascular endothelial cells play a critical role in vascular homeostasis and their function is influenced by oxygen tension, which varies in a spatial-temporal manner across tissues (Keeley & Mann, Physiol. Rev. 99:161-2340. This study investigates the effects of adapting human brain microvascular endothelial cells (hCMEC/D3) to physiological oxygen tension (5kPa O₂) on their basal potassium (K⁺) channel activity, as well as agonist- induced whole-cell K⁺ currents. Our data show that hCMEC/D3 exhibited only outwardly rectifying K⁺ currents. Compared to standard culture under 18 kPa O₂, long-term adaptation of hCMEC/D3 to 5 kPa O₂ exhibited larger outward currents under basal (+60mV: 0.598±0.058nA) and NO-stimulated whole-cell K⁺ currents (+60mV: 0.795±0.0.075nA), measured at –20mV and +60mV holding potentials using Nanion Port-a-Patch apparatus maintained in an oxygen-controlled Scitive dual workstation (Baker, USA).  Acute application of a NO donor (NOC-7, 400mM) increased outward currents only under 5kPa (40mV: P=0.0053 and 60mV: P<0.0001, two-way ANOVA). However, under 18kPa O₂, NO did not induce a current potentiation at any holding voltage (two-way ANOVA). To examine the effects of sequentially blocking BK, IK and SK channels, different K+ currents were pharmacologically isolated using tetraethylammonium (TEA, 10mM), TRAM-34 (5μM) and apamin (100nM) revealed differential effects in cells adapted to 5kPa or 18kPa O₂. Under 5kPa O₂ cells also exhibited a larger proportion of currents sensitive to all blockers (82%) compared to 44% in hyperoxic cultures. At 5kPa O₂, each inhibitor caused a significant reduction in whole-cell current amplitudes at 50mV relative to basal control currents (data expressed as normalised current relative to maximal amplitude at 50mV, 5kPa and 18kPa: TEA: 58±9% P=0.0291 and 74±7% P=0.2394, TEA+TRAM-34: 37±2% P=0.0011 and 65±14% P=0.0743, TEA+TRAM-34+apamin: 18±2% P<0.0001 and 56±12, P=0.0193, n=3 each, two-way ANOVA). Changes in pericellular O₂ levels had negligible effects on protein expression and fluorescence intensity of KCa1.1, KCa3.1 and KCa2.3 channels, suggesting that changes in whole-cell currents in were due to channel modulation. Thus, our findings reveal that physiological O₂ tension shapes the electrophysiological phenotype of human EC by modulating K⁺ channel function and NO responsiveness. The novel insights into the modulation of EC K⁺ channels by O₂ has implications for the regulation of vascular tone and design and use of experimental models in vitro for high throughput drug discovery and clinical translation.