Ischemic stroke is associated with a surge in reactive oxygen species generation during reperfusion. The narrow therapeutic window for the delivery of intravenous thrombolysis and endovascular thrombectomy limits therapeutic options for patients. Thus, understanding the mechanisms regulating neurovascular redox defenses are key for improved clinical translation. Our previous studies in a rodent model of ischemic stroke established that activation of Nrf2 defense enzymes by pretreatment with sulforaphane (SFN) affords protection against neurovascular and neurological deficits. We here further investigate SFN mediated protection in mouse brain microvascular endothelial cells (bEnd.3) adapted long-term (5 days) to hyperoxic (18 kPa) and normoxic (5 kPa) O₂ levels. Using an O₂-sensitive phosphorescent nanoparticle probe, we measured an intracellular O₂ level of 3.4 ± 0.1 kPa in bEnd 3 cells cultured under 5 kPa O₂. Induction of HO-1 and GCLM by SFN (2.5 μM) was significantly attenuated in cells adapted to 5 kPa O₂, despite nuclear accumulation of Nrf2. To simulate ischemic stroke, bEnd.3 cells were adapted to 18 or 5 kPa O₂ and subjected to hypoxia (1 kPa O₂, 1 h) 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 transcription 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 MitoSOX™ Red and FeRhoNox™-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 antioxidant therapies targeting the Keap1-Nrf2 defense pathway in treatment of ischemia-reperfusion injury in stroke, coronary and renal disease will require in vitro studies conducted under well-defined O₂ levels.