Background Endoscopic submucosal dissection (ESD) is accepted as one of the treatments for en bloc resection of large superficial colorectal lesions. This procedure is performed by using air insufflation, is time consuming, and is associated with severe abdominal discomfort. The safety and efficacy of carbon dioxide (CO 2) insufflation during colonoscopy already has been assessed in some trials. Objective To assess the safety and efficacy of CO 2 insufflation instead of air insufflation during colorectal ESD with the patient under conscious sedation. Design A case-control series with a historical control. Patients A total of 35 consecutive patients were enrolled in this study. Another 35 consecutive patients who previously received colorectal ESDs by using air insufflation were included as a historical control. Interventions Arterial partial pressure of CO 2 (pCO 2) was measured before and after each procedure with the total dose of midazolam used as an index of abdominal discomfort. Main Outcome Measurements and Results The mean (standard deviation [SD]) operation time was 90 ± 57 minutes in the CO 2 group and 100 ± 80 minutes in the control group (not significant). In the CO 2 group, the mean (SD) dose of midazolam was significantly lower than that of the control group; 5.6 ± 4.9 mg and 9.7 ± 5.9 mg, respectively ( P = .005). Blood analysis revealed a slight pCO 2 elevation in the CO 2 group; however, only 2 patients complained of mild abdominal discomfort. Limitations Abdominal discomfort and pCO 2 were not evaluated in the control group. Conclusions This study strongly suggests that CO 2 insufflation is safe and effective during lengthy colonic endoscopic procedures, eg, ESD, with the patient under conscious sedation.