Background: CO2 is rapidly absorbed from the colon and eliminated via the lung. Insufflation of CO2 instead of air during colonoscopy can reduce distention-induced pain. Objective: This study aimed to evaluate the effects of CO2 insufflation on pain during intubation and extubation and to identify predictors of pain and discomfort during colonoscope insertion. Design: Prospective, randomized, controlled trial. Setting: Single tertiary medical center in Taiwan. Patients: A total of 193 patients enrolled from September 2010 through June 2011. Interventions: Colonoscope insertion with either air or CO2 insufflation. CO 2 was used for extubation in both groups. Main Outcome Measurements: The main outcome measurement was pain, recorded on a 10-point visual analog scale (VAS) for left-sided colonoscope insertion and right-sided colonoscope insertion and at 1, 3, 6, and 24 hours post-procedure. Colonoscope cecal intubation time and extubation time, completeness of intubation, and loop formation were also assessed. Results: CO2 insufflation during colonoscope intubation was used in 98 patients and air in 97 patients. The mean pain scores during intubation were low (2-3) for patients undergoing air insufflation and were not reduced further in patients receiving CO2. A mean pain score of 0 was reported by both groups for all postprocedure time points. Multivariate analysis identified sex, loop formation of the sigmoid colon, time to reach the transverse colon, and requested sedation as factors that significantly affect VAS pain scores. Limitations: This study was limited in scope to a single medical center with experienced endoscopists. Conclusions: We detected no significant benefit to the use of CO2 insufflation compared with air insufflation during intubation for colonoscopy performed by experienced colonoscopists. The absence of postprocedure pain in both groups supports previous observations that CO2 insufflation during extubation is effective in reducing postprocedure pain. Female sex and loop formation were identified as key factors influencing pain scores on colonoscope insertion. (Clinical trial registration number: TSGHIRB-099-05-081.) © 2013 American Society for Gastrointestinal Endoscopy.