Few studies have evaluated the degree of pain, the amount of retained gas, and the safety of carbon dioxide (CO 2 ) insufflation in patients undergoing double-balloon enteroscopy (DBE).To clarify the usefulness and safety of CO 2 insufflation during DBE.Single-center, prospective, randomized, double-blind, controlled trial.University hospital.Forty eligible patients with small-bowel disease for whom DBE was indicated were randomized to a CO 2 insufflation (CO 2 ) group or an air insufflation (air) group by means of sealed envelopes.DBE with insufflation of CO 2 or air.Efficacy evaluation was based on the degree of pain as assessed by use of a visual analog scale (VAS) and the amount of residual gas retention within the small and large bowels on radiography. The safety of CO 2 insufflation was evaluated by arterial blood gas analysis.Significantly fewer patients in the CO 2 group had severe pain of ≥50 mm on the VAS during DBE than in the air group (P = .02). Significantly less gas was retained in the small bowel just after and at 3 hours after DBE in the CO 2 group than in the air group (P = .003, P = .01, respectively). There was significantly less residual gas retention in the large bowel at 3 hours after DBE in the CO 2 group than in the air group (P = .02). There was no significant difference in pre-DBE and post-DBE partial pressure of oxygen in the blood (PaO 2 ) and partial pressure of carbon dioxide in the blood (PaCO 2 ) between groups.Small sample size.CO 2 insufflation is a safe and useful procedure when performed during DBE.