Introduction: Endotracheal intubation is an essential skill in emergency medicine requiring technical proficiency and sufficient preparation for a safe procedure. In the Helicopter Emergency Medical Service (HEMS), it is common to intubate the patient who needs an advanced airway prior to take-off. In-flight-intubation (IFI) is avoided because it is considered difficult due to environmental limitations of space, communication, and vibration. In contrast, IFI may shorten the total prehospital time since the procedure is conducted during the flight. We tested whether IFI can be performed safely and shorten transportation time. Methods: We conducted a retrospective cohort study with patients transported from Apr 2010 to Mar 2017 in a single center. We included patients ≥ 18 years who received prehospital intubation and excluded patients with emergent intubation at the scene. We divided the observational cohort into two groups. The Flight group (FG): included patients intubated during the flight. The Ground group (GG): included patients intubated prior to take-off. HEMS crews transported both groups. The primary outcome was the proportion of successful intubations. Secondary outcomes included total prehospital time and the incidence of complications such as hypoxia and hypotension. Result: We analyzed 376 patients during the study period. There were 192 cases in FG and 184 cases in GG. Intubation success rate did not differ between the two groups (FG vs GG: 98.4% vs 97.3%, p = 0.50). There were no differences in hypoxia (FG vs GG: 3.4% vs 4.2%, p = 1.00) or hypotension (FG vs GG: 5.1% vs 5.3%, p = 1.00) between two groups. Scene time was shorter in FG (FG vs GG: 7 min vs 14 min, p < 0.001), as was total prehospital time (FG vs GG: 33.5 min vs 40.0 min, p < 0.001). Conclusions: In-flight-intubation during HEMS could be safely performed without additional hypoxia or hypotension. In-flight-intubation by experienced providers shortened transportation time by an average of 7 minutes.