We recently reported that the cavitary 2-layer method (cTLM) allowed stable 24-hour preservation of canine intestine. The aim of this study was to examine the possibility of the 40-hour preservation by cTLM that is supplemented with glutamine. Canine jejunal segments (40 cm) were allotransplanted heterotopically without storage (group 1), after 40-hour cold storage with University of Wisconsin solution (group 2), cTLM (group 3), UW with 2% glutamine (group 4), or cTLM with 2% glutamine (group 5). Mucosal glutamine, histidine, tryptophan, glutathione, and adenosine triphosphate concentrations were determined immediately after preservation. At day 7 after the transplantation, maltose absorption test and histopathologic analysis were performed. Mucosal glutamine concentrations increased significantly with glutamine supplementation during preservation in groups 4 and 5 (P < .01). Mucosal adenosine triphosphate levels in cTLM groups (groups 3 and 5) were similar to those in group 1; group 4 showed very low levels after preservation, despite glutamine supplementation. Mucosal glutathione did not differ among groups 2, 3, 4, and 5 and were significantly lower than in group 1. In the absorption test, serum glucose curves showed a peak level by 30 minutes in groups 1 and 5; delayed peaks were seen in groups 2, 3, and 4 compared with group 1. The villous heights were 879, 555, 685, 688, and 773 microm in groups 1, 2, 3, 4, and 5, respectively (group 1 vs group 2, 3, and 4: P < .05). cTLM that was supplemented with glutamine extended a safe preservation period up to 40 hours in canine segmental small intestinal transplantation, although this study failed to elucidate a precise mechanism of the glutamine beneficial effect on the graft mucosa.