Conventional in vitro models classically use cells cultured in ambient air/5% CO2 which are hyperoxic compared to the environmental milieu in vivo. Given the intrinsic role of molecular O2 in determining reactive oxygen species (ROS) production and redox homeostasis, the current study sought to determine whether redox homeostasis is altered in cells adapted to physiological O2 tensions. Experiments were conducted using primary human umbilical vein endothelial cells (HUVEC) cultured under conventional 21% O2 or adapted to physiological 5% O2 for 1-day or 5-days. Using specialised oxygen workstations, we assessed (i) transcriptional DNA binding activity of the key redox-sensitive transcription factor Nrf2 by ELISA, (ii) induction of Nrf2-mediated antioxidant and phase II enzymes, GSH related genes or Nrf2 negative regulatory proteins Keap1 and Bach1 mRNA and protein by qPCR or immunoblotting, (iii) GSH synthesis by OPA fluorescence and (iv) cellular and mitochondrial ROS generation using DHE fluorescence. Whilst Nrf2 nuclear translocation or DNA binding of Nrf2 was not altered under 5% O2 conditions, progressive adaptation of cells to normoxic 5% O2 resulted in a depression of Nrf2-mediated antioxidant heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase (NQO1) induction in response to the known electrophilic Nrf2 inducer diethylmaleate. In contrast, GSH synthesis or induction of critical GSH related genes glutamate cysteine ligase catalytic subunit (GCLC) or the cystine glutamate transporter (xCT) were unaltered, whereas induction of the negative Nrf2 regulator Bach1, but not Keap1, was elevated under normoxic conditions. Measurement of cellular ROS generation using DHE or mitochondrial targeted DHE (Mitosox Red), revealed that adaptation of cells to normoxic 5% O2 results in alterations in ROS output with enhanced sensitivity to the mitochondrial complex I inhibitor antimycin A. Collectively these findings suggest that under normoxic 5% O2 conditions human endothelial cells exhibit a dramatically altered redox profile to cells cultured under standard air/5% CO2 conditions. Differential Nrf2 regulation of HO-1, GCLC and Bach1 suggests that undetermined factors modulated by O2 tension influence Nrf2 gene transcription, with significant implications for cellular ROS generation and ultimately cell redox homeostasis.