Purpose Primary human corneal endothelial cells (HCEnCs) cultured in room air are exposed to significantly higher O 2 concentrations [O 2 ] than what is normally present in the eye. We evaluated the growth and metabolism of HCEnCs cultured under physiological [O 2 ] (2.5%; [O 2 ] 2.5 ) and room air ([O 2 ] A ). Methods Primary cultures of HCEnCs from normal donors and donors with Fuchs dystrophy were grown at [O 2 ] 2.5 and [O 2 ] A . Growth and morphology were compared using phase-contrast microscopy, zonula occludens (ZO-1) localization, cell density measurements, and senescence marker staining. CD44 (cell quality) and HIF-1α (hypoxia-inducible factor-1α) levels were evaluated by Western blotting. Cell adaptability to a reversal of [O 2 ] growth conditions was measured with cell viability assays, and cell metabolism was assessed via oxygen consumption and extracellular acidification rates. Results HCEnCs grown at [O 2 ] A and [O 2 ] 2.5 displayed similar morphologies, ZO-1 localization, CD44 expression, and senescence. Cells from donors with Fuchs dystrophy grew better at [O 2 ] 2.5 than at [O 2 ] A . HIF-1α was undetectable. Cells displayed greater viability at [O 2 ] 2.5 than at [O 2 ] A . HCEnCs showed significantly greater proton leak ( P < 0.01), nonmitochondrial oxygen consumption ( P < 0.01), and spare capacity ( P < 0.05) for oxygen consumption rates, and greater basal glycolysis ( P < 0.05) with a decreased glycolytic reserve capacity ( P < 0.05) for extracellular acidification rates. Conclusions Primary HCEnCs show unique metabolic characteristics at physiologic [O 2 ]. The effect of [O 2 ] for optimization of HCEnC culture conditions should be considered. Translational Relevance With the advance of cell-based therapeutics for corneal endothelial diseases, [O 2 ] should be considered an important variable in the optimization of HCEnC culture conditions.