This study investigated the effect of glutamine (GLN) on expressions of small intestinal intraepithelial lymphocyte (IEL) gamma delta T-cell proinflammatory cytokines and apoptotic regulatory factor genes in a mouse model of hindlimb ischemia/reperfusion (IR) injury. Mice were assigned to a normal control group and three IR groups. Mice in the normal control group received no ischemia treatment, whereas IR groups had hindlimb ischemia for 90 min with subsequent 0 (IR0) or 24 h (IR24) of reperfusion. The IR0 group was sacrificed immediately after reperfusion. The IR24S group was injected with saline, and the IR24G group was given 0.75 g GLN/kg of body weight once via a tail vein before reperfusion. The IR24 groups were sacrificed 24 h after reperfusion. Small intestinal IEL gamma delta T cells of the animals were isolated for further analysis. Results showed that IR injury resulted in lower small intestinal IEL gamma delta T-cell percentages and higher proinflammatory cytokine messenger RNA expressions of interleukin-1 beta (IL-1 beta), IL-6, and tumor necrosis factor-alpha by IEL gamma delta T cells. Compared with the IR24S group, the IR24G group had a higher IEL gamma delta T-cell percentage. Multiples of change of messenger RNA of proliferation gene expressions of the antiapoptotic Bcl-xl (B-cell lymphoma-extra large) and IL-7 receptor in the IR24G group were higher, whereas expressions of the keratinocyte growth factor and bacterial lectin regenerating islet-derived (Reg)III gamma were lower in IEL gamma delta T cells. Histological findings also showed that damage to the intestinal mucosa was less severe in the IR group with GLN. These results indicated that a single dose of GLN administered before reperfusion maintained small intestinal IEL gamma delta T cell populations and reduced expressions of intestinal inflammatory cytokines, which may have consequently ameliorated the severity of IR-induced small intestinal epithelial injury.