This study investigated the effect of glutamine (GLN) on expressions of small intestinal intraepithelial lymphocyte (IEL) γδT-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 γδT cells of the animals were isolated for further analysis. Results showed that IR injury resulted in lower small intestinal IEL γδT-cell percentages and higher proinflammatory cytokine messenger RNA expressions of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α by IEL γδT cells. Compared with the IR24S group, the IR24G group had a higher IEL γδT-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)IIIγ were lower in IEL γδT 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 γδT cell populations and reduced expressions of intestinal inflammatory cytokines, which may have consequently ameliorated the severity of IR-induced small intestinal epithelial injury.