Introduction. Adverse hemodynamic effects with severe bradycardia have been previously reported during positive pressure insufflation of the right thoracic cavity in humans. To the best of our knowledge, this is the first report of asystole during thoracoscopic surgery with positive pressure insufflation. Case presentation. A 63-year-old Caucasian woman developed asystole at the onset of positive pressure insufflation of her right hemithorax during a thoracoscopic single-lung ventilation procedure. Immediate deflation of pleural cavity, intravenous glycopyrrolate and atropine administration returned her heart rhythm to normal sinus rhythm. The surgery proceeded in the absence of positive pressure insufflation without any further complications. Conclusions: We discuss the proposed mechanisms of hemodynamic instability with positive pressure thoracic insufflation, and anesthetic and insufflation techniques that decrease the likelihood of adverse hemodynamic events.