We previously extablished in a rodent model of ischemic stroke that activation of NRF2 defenses by pretreatment with sulforaphane (SFN) affords protection against neurovascular and neurological deficits. To further investigate the molecular mechanisms, we further investigated SFN-mediated protection in mouse brain microvascular endothelial cells (bEnd.3) adapted long-term to hyperoxic (18 kPa), normoxic (5 kPa) or hypoxic (1 kPa) O2 levels. Using an O2-sensitive phosphorescent nanoparticle probe, we measured an intracellular O2 level of 3.4 ± 0.1 kPa in bEnd3 cells cultured under 5 kPa O2. Induction of HO-1 and GCLM by SFN (2.5 µM) was significantly attenuated in cells under 5 kPa O2, despite nuclear accumulation of Nrf2. To simulate ischemic stroke, cells were adapted to 18 or 5 kPa O2 and subjected to hypoxia (1 kPa O2, 1h) and reoxygenation. In cells adapted to 18 kPa O2, reoxygenation induced free radical generation was abrogated by PEG-SOD and significantly attenuated by pretreatment with SFN (2.5 µM). Silencing NRF2 abrogated HO-1 and NQO1 induction and led to a significant increase in reoxygenation induced free radical generation. Notably, reoxygenation induced oxidative stress, assayed using the luminescence probe L-012 and fluorescence probes MitoSOXTM Red and FeRhoNoxTM-1, was diminished in cells cultured under 5 kPa O2, indicating an altered redox phenotype in brain microvascular cells adapted to physiological normoxia. As redox and other intracellular signaling pathways are critically affected by O2, the development of high throughput therapies targeting KEAP1-NRF2 in the treatment of reperfusion injury in stroke, coronary and renal disease will require the design of in vitro studies to recapitulate the redox phenotype of cells in vivo (see Keeley & Mann, Physiol. Rev. 2019;99(1):161-2340).