One of the methods used to reduce pain and discomfort during colonoscopy is insufflation of carbon dioxide instead of air. However, the actual benefit of carbon dioxide insufflation is not unequivocally proven. The aim of the study was to evaluate the advantages of carbon dioxide insufflation during screening colonoscopy. A total of 200 patients undergoing screening colonoscopy between 2010 and 2011 were included in the prospective, randomized study carried out in a surgical referral center. Screening unsedated colonoscopy with either air or carbon dioxide insufflation was performed; patients were randomly assigned to air or carbon dioxide group by means of computer-generated randomization lists. All examinations were performed in an ambulatory setting with standard videocolonoscopes. The main outcomes analyzed were (a) duration of the entire procedure, (b) cecal intubation time, and (c) pain severity immediately, 15, and 60 min after the procedure. Group I included 59 women and 41 men and group II included 51 women and 49 men. The duration of the procedure was circa 10 min in both groups. Pain score values immediately and 15 min after the procedure were similar in both groups (P=0.624 and 0.305, respectively). A lower pain score was observed only after 60 min in patients insufflated with carbon dioxide (1.28 vs. 1.54, P=0.008). No pain reduction was observed in women and in obese patients (BMI>30). Carbon dioxide insufflation during unsedated screening colonoscopy does not decrease the duration of the procedure and appears to reduce pain intensity at 60 min after examination to an extent without clinical significance. The study was registered at ClinicalTrials.gov, number NCT01461564.