Background: Hypoxemia is a frequent adverse event occurring during flexible bronchoscopy and is usually prevented by close monitoring and, if needed, oxygen supplementation by nasal cannula. Objective: We aim to demonstrate that tracheal oxygen supplementation during flexible bronchoscopy is a feasible, safe and effective method to restore oxygen saturation levels after oxygen desaturation. Methods: In a first phase, we compare oxygen supplementation by the tracheal or nasal route in a single blinded cross-over design in healthy volunteers. In a second phase, we study patients referred for diagnostic flexible bronchoscopy, who desaturate despite oxygen supplementation by nasal cannula, in order to assess the ability to correct hypoxemia through tracheal oxygen supplementation. Results: In the first phase, the mean capillary partial pressure of oxygen was 181 mm Hg when oxygen at a flow rate of 4 liters/min was administered by the tracheal route, compared to 125 mm Hg by the nasal route (p < 0.001). The capillary partial pressure of carbon dioxide was not significantly different. During 950 bronchoscopic procedures in the second phase of the trial, 30 patients desaturated below 90% despite oxygen supplementation by nasal cannula. In 22 out of these 30 patients, switching to the tracheal route resulted in a correction of the saturation within 120 s. In the remaining 8 patients, saturation levels were corrected after increasing the oxygen flow rate to 4 liters/min. After restoring saturation levels, the bronchoscopy could be completed in 25 of 30 patients. Conclusions: Tracheal oxygen supplementation is safe, feasible and an effective way to restore oxygen saturation levels during flexible bronchoscopy.