INTRODUCTION: Technical difficulty and unfamiliar surgical anatomy are the main challenges in transanal total mesorectal excision. Precise 3-dimensional real-time image guidance may facilitate the safety, accuracy, and efficiency of transanal total mesorectal excision. TECHNIQUE: A preoperative CT was obtained with 10 skin fiducials and further processed to emphasize the border of the anatomical structure by 3-dimensional modeling and pelvic organ segmentation. A forced sacral tilt by placing a 10-degree wedge under the patient's sacrum was induced to minimize pelvic organ movement caused by lithotomy position. An optical navigation system with cranial software was used. Preoperative CT images were loaded into the navigation system, and patient tracker was mounted onto the iliac bone. Once the patient-to-image paired point registration using skin fiducials was completed, the laparoscopic instrument mounted with instrument tracker was calibrated for instrument tracking. After validating the experimental setup and process of registration by navigating laparoscopic anterior resection, stereotactic navigation for transanal total mesorectal excision was performed in the low rectal neuroendocrine tumor. RESULTS: The fiducial registration error was 1.7 mm. The accuracy of target positioning was sufficient at less than 3 mm (1.8 ± 0.9 mm). Qualitative assessment using a Likert scale was well matched between the 2 observers. Of the 20 scores, 19 were judged as 4 (very good) or 5 (excellent). There was no statistical difference between mean Likert scales of the abdominal or transanal landmarks (4.4 ± 0.5 vs 4.3 ± 1.0, p = 0.965). CONCLUSIONS: Application of an existing navigation system to transanal total mesorectal excision for a low rectal tumor is feasible. The acceptable accuracy of target positioning justifies its clinical use. Further research is needed to prove the clinical need for the procedure and its impact on clinical outcomes.