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Abstract In oxygen (O₂)‐controlled cell culture, an indispensable tool in biological research, it is presumed that the incubator setpoint equals the O₂tension experienced by cells (i.e., pericellular O₂). However, it is discovered that physioxic (5% O₂) and hypoxic (1% O₂) setpoints regularly induce anoxic (0% O₂) pericellular tensions in both adherent and suspension cell cultures. Electron transport chain inhibition ablates this effect, indicating that cellular O₂consumption is the driving factor. RNA‐seq analysis revealed that primary human hepatocytes cultured in physioxia experience ischemia‐reperfusion injury due to cellular O₂consumption. A reaction‐diffusion model is developed to predict pericellular O₂tension a priori, demonstrating that the effect of cellular O₂consumption has the greatest impact in smaller volume culture vessels. By controlling pericellular O₂tension in cell culture, it is found that hypoxia vs. anoxia induce distinct breast cancer transcriptomic and translational responses, including modulation of the hypoxia‐inducible factor (HIF) pathway and metabolic reprogramming. Collectively, these findings indicate that breast cancer cells respond non‐monotonically to low O₂, suggesting that anoxic cell culture is not suitable for modeling hypoxia. Furthermore, it is shown that controlling atmospheric O₂tension in cell culture incubators is insufficient to regulate O₂in cell culture, thus introducing the concept of pericellular O₂‐controlled cell culture.