To compare the effectiveness and safety of transgastric and transrectal pure natural orifice transluminal endoscopic surgery (NOTES) for cholecystolithotomy.This was a single-center retrospective comparative study of consecutive patients who underwent pure NOTES for either transrectal or transgastric gallbladder-preserving cholecystolithotomy between September 2017 and April 2020. Patients with symptomatic cholelithiasis were assigned for transrectal or transgastric NOTES based on the patients' choice. Treatment success, postoperative pain, peritonitis, time to resume normal diet, and duration of hospitalization were compared.The technical success rate was 100%. Forty-eight patients underwent successful NOTES cholecystolithotomy via the transrectal (n = 26) or transgastric route (n = 22). One (3.8%) patient in the transrectal NOTES group experienced postoperative abdominal pain compared to 6 (27.3%) in the transgastric NOTES group (P = 0.04). Fever and bile peritonitis developed in one (3.8%) patient in the transrectal NOTES group versus 8 (36.4%) in the transgastric NOTES group (P = 0.005). A postoperative fluid diet was commenced at 6 h with the transrectal approach versus on day 3 for the transgastric NOTES group. The mean postoperative hospitalization for transrectal and transgastric NOTES groups was 4.5 days versus 7 days (P = 0.001). Three patients in the transgastric NOTES group developed postoperative gastric fistula.Transrectal NOTES has advantages over transgastric NOTES, including preserved spatial orientation, relatively easier removal of specimens, early food intake, shorter hospitalization, fewer postoperative complications and less pain. Multicenter clinical trials with long-term follow-up are needed to confirm the safety and efficacy of both approaches.