Fuchs endothelial corneal dystrophy (FECD) results from genetic and environmental factors triggering mitochondrial and oxidative stress in corneal endothelial cells (CEnCs) leading to CEnC death and corneal opacification. FECD is more common in women than men, but the basis for this observation is unknown. Because FECD is commonly diagnosed around the time of the menopausal transition in women when estrogen levels decrease precipitously, we studied the effects of the potent estrogen,17-β estradiol (E2) on growth, oxidative stress, and metabolism in primary cultures of human CEnCs (HCEnCs) under conditions of physiologic 2.5% O2([O2]2.5) and under hyperoxic stress ([O2]A: room air + 5% CO2). We hypothesized that E2 would counter the stresses of the hyperoxic environment in HCEnCs. HCEnCs were treated ± 10 nM E2 for 7–10 days at [O2]2.5and [O2]Afollowed by measurements of cell density, viability, reactive oxygen species (ROS), mitochondrial morphology, oxidative DNA damage, ATP levels, mitochondrial respiration (O2consumption rate [OCR]), and glycolysis (extracellular acidification rate [ECAR]). There were no significant changes in HCEnC density, viability, ROS levels, oxidative DNA damage, OCR, and ECAR in response to E2 under either O2condition. We found that E2 disrupted mitochondrial morphology in HCEnCs from female donors but not male donors at the [O2]Acondition. ATP levels were significantly higher at [O2]2.5compared to [O2]Ain HCEnCs from female donors only, but were not affected by E2. Our findings demonstrate the overall resilience of primary HCEnCs against hyperoxic stress. The selective detrimental effects of hyperoxia and estradiol on HCEnCs from female but not male donors suggests mechanisms of toxicity based upon cell-sex in addition to hormonal environment.