Culture of human endothelial cells is routinely undertaken under atmospheric oxygen conditions (18-20% O2), whereas oxygen levels in vivo are typically ~3-5% within most vascular beds (Ward, 2008). We have previously used an O2-regulated workstation and plate reader to characterise the phenotype of human endothelial cells under defined O2 concentrations and, using an O2 sensitive nanoparticle probe MitoXpress-INTRA, confirmed an intracellular O2 content of 3.6% in HUVEC cultured long-term under physiological (5%) O2 levels (Chapple et al., 2016). As nitric oxide (NO) signalling is intimately regulated by O2 levels, we here investigate NO signalling in human umbilical vein endothelial cells (HUVEC) cultured long-term under 18% or physiological (5%) O2 levels and then stimulated with the inflammatory mediator histamine or physiological shear stress. Culture of HUVEC under 5% O2 (5 days) significantly decreased histamine (10 µM, 5 min), but not shear stress (15 dynes/cm2, 10 min), stimulated eNOS phosphorylation (3.1- vs 1.8-fold, n=6, P<0.01 2-way ANOVA) and eNOS activity (2.5±0.3 vs 1.6±0.1, mean ± S.E.M., n=4, P<0.05 2-way ANOVA). Despite this, histamine-stimulated cGMP production was unaffected (6.5±1.3 vs 6.5±1.2 pmol/mg, n=6), and exposure to shear stress elicited a significantly greater cGMP production under 5% O2 (5.8±0.7 vs 13.4±2.6 pmol/mg, n=9, P<0.001). Higher NO bioavailability under physiological O2, investigated using mathematical modeling and in vitro experiments with the NO donor DETA NONOate, could account for the disparity between eNOS activity and cGMP production. Treatment with okadaic acid (100 nM, 30 min), a selective inhibitor of protein phosphatase 2A (PP2A), reversed the affects of adaptation to 5% O2 on histamine-stimulated eNOS phosphorylation and significantly increased cGMP production (4.9±0.8 vs 10.1±3 pmol/mg, n=5, P<0.001 2-way ANOVA). Increased basal microsomal association of PP2A under 5% O2 (14.2±1.3 vs 28.9±3.4%, n=5, P<0.05 2-way ANOVA), facilitated a more rapid interaction with eNOS, assessed by in situ proximity ligation, and therefore accelerated dephosphorylation. This study provides the first evidence that long-term culture of human endothelial cells under physiologically relevant O2 levels significantly alters their phenotype and responses to select vasoactive stimuli. We conclude that culture of endothelial cells under hyperoxic (18% O2) conditions induces an oxidative, inflammatory phenotype that may prime them towards inflammatory stimulation, possibly via delayed targeting of eNOS by PP2A. These novel insights into endothelial physiology in vitro highlight their sensitivity to O2, and the consequences associated with fluctuations away from physiological normoxia (~3-5%).