Purpose: We conducted a prospective study to evaluate the effect of CO2 pneumoperitoneum and increased intra-abdominal pressure on arterial blood gases, end-tidal CO2 (ETCO2), nitric oxide (NO), blood and tissue malondialdehyde (MDA), and total antioxidant (TAOx) levels during laparoscopic cholecystectomy. Methods: Fifty selected patients with cholelithiasis were randomized to undergo either laparoscopic or open surgery. Blood samples were taken pre-, mid-, and postinsufflation, and 24 h postoperatively. To determine the tissue MDA level, tissue samples were taken from the gallbladder just after removal. Results: The increased levels of ETCO2 and PCO2, caused by CO2 pneumoperitoneum resulted in a minimal decrease in blood pH during the laparoscopic surgery. Although low levels of blood MDA were seen 30 min after the start of laparoscopy, due to less oxidative stress response and tissue trauma, increased levels of tissue MDA levels indicated that the gallbladder was more traumatized during laparoscopic dissection and handling. NO levels were slightly lower in the laparoscopic cholecystectomy (LC) group, but there were no significant differences compared with the open cholecystectomy group (OC). TAOx levels were similar in both groups 30 min after the start the procedure, but were much lower in the LC group 24 h postoperatively. Conclusions: These findings suggest that the antioxidant defense system is stimulated less with less oxidative stress, providing further evidence to support the opinion that LC is a safe technique.