Background and AimAlthough natural orifice transluminal endoscopic surgery (NOTES) has gained in popularity, the preferred route of access to the peritoneal cavity is still debated. Here, we compare and discuss the safety and effectiveness of trans‐gastric and trans‐rectal pure NOTES cholecystolithotomy.Patients and MethodsThis was a single center retrospective comparative study of consecutive patients who underwent either pure NOTES trans‐rectal or trans‐gastric gallbladder preserving cholecystolithotomy at the First Affiliated Hospital of Zhengzhou University between September 2017 and April 2020. Patients with symptomatic cholelithiasis were either selected for trans‐rectal or trans‐gastric NOTES based on the patients’ choice. The two methods of access were compared with regard to treatment success, post‐operative pain, peritonitis, time to normal diet intake and duration of hospital stay.ResultsThe procedure technical success rate was 100%. 48 patients underwent successful NOTES cholecystolithotomy (trans‐rectal, n=26 and trans‐gastric, n=22). One (3.8%) patient in trans‐rectal group experienced postoperative abdominal pain compared to 6 (27%) patients in the trans‐gastric group (p=0.04). Fever and bile peritonitis developed in 1 (4%) in trans‐rectal patient vs. 8 (36%) trans‐gastric patients (p=0.005). A fluid diet was commenced at 6 hours with the trans‐rectal approach vs. on day 3 for trans‐gastric patients. The mean post‐operative hospital stay for trans‐rectal patients was 4.5 days vs.7 days for trans‐gastric patients (p=0.031). Three (14%) trans‐gastric patients developed gastric fistula post‐operatively.ConclusionTrans‐rectal NOTES has advantages over trans‐gastric NOTES including preserved spatial orientation, a larger port for introduction of instruments and removal of specimen, early food intake, shorter hospital stay, fewer post‐operative complications and less pain. Further studies especially prospective multi‐center clinical trials with long term follow up are needed to confirm the safety and efficacy of both approaches.This article is protected by copyright. All rights reserved.