We previously developed the two-layer cold storage method (TLM), which allows sufficient oxygen delivery to the canine pancreas during preservation, and successfully achieved 96-h preservation. In this study, we applied a modified TLM (cavitary TLM) to small bowel preservation in a canine heterotopic transplant model. Using simple storage in University of Wisconsin solution (UWM, group 1, n = 12) or cavitary TLM (group 2, n = 8), 40 cm segments of the jejunum were preserved for 24 h at 4 degrees C. The nonpreservation group served as the control (group 3, n = 8). The grafts were implanted heterotopically as a Thiry-Vella loop. Eleven of 12 dogs in group 1 died within 3 days post-transplant as a result of graft intraluminal hemorrhage, while all dogs in groups 2 and 3 survived until day 7. Histological analyses showed almost normal structures of the graft mucosa in groups 2 and 3 at day 7. Results from maltose and acetaminophen absorption tests in group 2 were comparable to those in group 3. Only one survivor in group 1 showed distinct graft mucosal damage, confirmed by histological and functional analyses. In our transplant model, the canine small bowel was successfully preserved by cavitary TLM for at least 24 h, while this preservation time was beyond the limit with UWM.