OBJECTIVE: To determine ideal insufflation pressures during transanal minimally invasive surgery (TAMIS) in canine cadavers for rectal submucosal transection and incisional closure. ANIMALS: 16 canine cadavers. PROCEDURES: Cadavers were placed in lateral recumbency. Urinary catheters were placed to measure intra-abdominal pressure (IAP). A single access port was placed to establish a pneumorectum. Cadavers were placed in insufflation groups of 6 mmHg to 8 mmHg (group 1), 10 mmHg to 12 mmHg (group 2), or 14 mmHg to 16 mmHg (group 3). Defects in the rectal submucosa were created and closed with a unidirectional barbed suture. Duration for each procedure and subjective ease of identifying the transection plane and performing incisional closure were assessed. RESULTS: The single access port was successfully placed in dogs weighing 22.7 kg to 48 kg. The ease of each step of the procedure was not influenced by the insufflation pressure. The median surgical duration for group 1 was 740 seconds (range = 564 to 951 seconds), 879 seconds (range = 678 to 991 seconds) for group 2, and 749 seconds (range = 630 to 1,244 seconds) for group 3 (P = .650). The insufflation pressure increased the IAP (P = .007). Perforation of the rectum happened in 2 cadavers in group 3. CLINICAL RELEVANCE: The duration of each step of the procedure was not significantly influenced by insufflation pressure. Defining the dissection plane and performing resection was more challenging in the highest-pressure group. Rectal perforation occurred only with the 14 mmHg to 16 mmHg insufflation pressure. Single access port usage with TAMIS may provide a readily available, minimally invasive approach for the resection of rectal tumors in dogs.