Despite pressurization of airliner cabins, some passengers experience in-flight hypobaric hypoxia with blood oxygen saturation dropping below 90%, potentially causing discomfort and increasing the risk of medical events. Enrichment of the cabin air with CO2 may augment passengers’ blood and tissue oxygenation by stimulating respiratory drive, thereby increasing health and safety during air travel. In a randomized double-blind crossover study we exposed 17 healthy adults (8 women; age range: 18-40 years) on separate days to two ambient CO2 levels (0.1 vs. 1.0% indoor sea level equivalents; 0.76 vs. 7.60 mmHg partial pressure) during 6 hours of hypobaric hypoxia (~565 mmHg total barometric pressure; corresponding to 2,438 m altitude) in an altitude chamber simulating long-haul flight conditions. We measured oxygen saturation of the blood (SpO2), brain and muscle (tissue saturation index derived from near-infrared spectroscopy), respiration, and cognitive function (sustained attention, working memory, hand-eye coordination) hourly. Additionally, we conducted capillary blood gas analyses at baseline, 15 minutes and 6 hours after hypoxia onset. During hypobaric hypoxia ambient CO2 enrichment on average increased pCO2 from 36.3 to 38.3 mmHg, pO2 from 60.9 to 68.3 mmHg, and minute ventilation from 9.7 to 10.4 l/min, while reducing the time fraction of SpO2 textless 90% from 18.8 to 2.5%. Tissue oxygenation increased in the brain from 62.8 to 63.9% whereas no change was found in muscle. High ambient CO2 had no effect on cognitive performance. Taken together, enrichment of cabin air with CO2 during hypobaric hypoxia may improve blood and brain oxygenation.