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Purpose: The optimal approach for NOTES is unknown. We conducted a prospective, single-center, non-randomized comparison of transgastric (TG) versus transvaginal (TV) hybrid NOTES cholecystectomy in humans. We report our intra-operative and short-term clinical outcomes.Methods: Patients requiring cholecystectomy were enrolled in an IRB approved protocol at a single center. Exclusion criteria included body mass index (BMI) >40 kg/m2, gallstone>1.5cm, choledocholithiasis, acute cholecystitis, esophageal stricture, prior pelvic or gastric surgery, extensive endometriosis, active infection, pregnancy, and contraindication to laparoscopy. Data were collected prospectively. All operations were performed using standard dual channel endoscopes with at least one 5mm laparoscopic port for assistance. Intra-operative measurements included total operative time, time for peritoneal access, dissection time, time to close the access site, estimated blood loss (EBL), and number of laparoscopic ports used. Length of stay (LOS) and standardized VAS (1-10) pain scores were tracked postoperatively. Intra- and post-operative complications were noted and reported to the appropriate IRB.Results: Nine patients underwent either TG (N=4) or TV (N=5) hybrid cholecystectomies. Mean age ± standard deviation (47 ± 12 v. 35 ± 2 yrs), BMI (30 ± 5 v. 29 ± 4 kg/m2), and history of previous abdominal surgery (n=1 v. n=1) were not significantly different between TG and TV groups, respectively. TG procedures resulted in longer total operative times (323 ± 58 v. 165 ± 33 min, p =0.014), peritoneal access times (93 ± 48 v. 19 ± 6 min, p= 0.014), access closure times (63 ± 13 v. 9 ±4 minutes, p =0.014), and LOS (52 ± 36 v. 14 ± 10 hrs, p = 0.027) compared to TV procedures. EBL (88 ± 25 v. 80 ± 102 mL), number of laparoscopic ports (2.5 ± 0.6 v. 1.6 ± 0.5), dissection time (110 ± 21 v. 99 ± 25 minutes), post-operative day 1 pain score (4.8 ± 2.1 v. 2.2 ± 1.9), and total number of complications (n=1 v. n=1) were similar between TG and TV groups, respectively. One TG patient had significant abdominal pain on POD1 and underwent repeat laparoscopy. This patient was found to have mild pancreatitis but no gastrotomy leak. One TV patient had mild intraoperative hematuria and underwent rigid cystoscopy, which revealed a urethral abrasion from foley catheter trauma that resolved spontaneously.Conclusion: Hybrid NOTES cholecystectomy is safe and feasible using either a TG or TV approach. The TV approach is technically easier to perform, and allows a shorter total operative time, access time, closure time, and length of stay compared with a TG approach.