Intestinal mucosal damage and bacterial translocation are clinical problems that may be caused by the use of ionizing radiation. Glutamine (GIn) support reduces the mucosal barrier in several ways. This study was undertaken to investigate the effect of timing of GIn-enriched enteral nutrition (EN) on bacterial translocation and mucosal damage due to radiotherapy (RT). A rat model of whole body irradiation was designed in which a single dose of 485 cGy was given. A total of 50 rats were randomly assigned to the following 5 groups, each of which comprised 10 rats: (1) balanced rat chow given for 8 days without RT (group 1); (2) balanced rat chow given 4 days before and 4 days after RT (group 2); (3) GIn-enriched EN given 4 days before RT (group 3); (4) GIn-enriched EN given 4 days after RT (group 4); and (5) GIn-enriched EN given 4 days before and 4 days after RT (group 5). Mesenteric lymph node and ileum samples were removed for evaluation of bacterial translocation (BT) and histopathologic investigation, respectively. BT and intestinal mucosal injury scores in all rats that received RT were higher than in rats without RT. No difference was seen in parameters between groups 3 and 4 (P >.05, P >.016, respectively); BT and intestinal mucosal injury scores of group 5 were significantly lower than those of groups 3 and 4 (P <.05, P <.016, respectively). Meanwhile, the BT and mesenteric injury scores of group 5 were significantly lower than those of group 2 (P <.05, P <.01 6, respectively). As a result, intestinal injury due to RT was significantly decreased by GIn-enriched EN support given before and after whole body RT.