Objective To investigate the effect of glutamine in combination with umbilical cord blood mesenchymal stem cells (MSCs) transplantation on intestinal ischemia-reperfusion injury in rats. Methods Umbilical cord blood mesenchymal stem cells were isolated, and were labeled with CM-DiI fluorescent dye. Eighty Sprague-Dawley rats were randomly divided into normal control group, ischemia reperfusion injury group, glutamine group, MSCs transplantation group and combined group with 15 rats in each group. The control group received saline enema. The injury group was treated with TNBS (ethanol dilution) enema. The glutamine group at 1 h after TNBS received intravenous injection of 0.45 g/kg glutamine. The rats of MSCs transplantation group had tail vein injection of 1 ×10¹⁰/L umbilical cord blood mesenchymal stem cell suspension, and the combined group received intravenous injection of glutamine 0.45 g/kg and 1×10¹⁰/L umbilical cord blood mesenchymal stem cell suspension. ELISA was used to detect the midgut fatty acid binding protein (iFABP), interleukin 6 (IL-6), and superoxide dismutase (SOD) content in the rat serum. The water content of intestinal tissue was detected at 1 h and 3 h after reperfusion in each group. The expressions of NF-kB, Bcl-2 and caspase-3 mRNA and proteins in the rat intestinal epithelial cells after treated with glutamine in combination with MSCs were detected by RT-PCR and Western blot assays. Results The fluorescent tracer method revealed that the transplanted MSCs cells were distributed in the intestinal mucosal lymphoid tissues and glandular epithelial cells, indicating that MSCs might be involved in the repair process of intestinal ischemia-reperfusion injury. The content of serum IFABP and IL-6 in the injured group was significantly higher than that in the control group, while significantly reduced in the glutamine group, MSCs transplantation group and combined group, with the most obvious in the combined group. The content of SOD in the injury group was significantly lower than that in the control group, and significantly increased than that in the glutamine group, MSCs transplantation group, with the most striking in the combined group (P<0.05 for all). The water content of intestinal tissue in the injury group at 1 and 3 hours after reperfusion was significantly higher than that in the control group, significantly lower in the glutamine group, MSCs transplantation group and the combined group, with the most decreased in the combination group, and there was no significant difference between the glutamine group and MSCs transplantation group (P>0.05). Compared with the control group, the caspase-3 and NF-kB mRNA and protein expressions in the intestinal mucosal epithelial cells of the injury group were significantly increased, and the expressions of Bcl-2 mRNA and protein were significantly reduced (P<0.05), the expressions of caspase-3 and NF-kB mRNA and protein were significantly reduced in the glutamine group, MSCs transplantation group and combined group. The expressions of Bcl-2 mRNA and protein were significantly increased (P<0.05), while no significant difference was shown between the glutamine group and MSCs transplantation group (P>0.05), but there was a significant difference between these two groups and the combined group (P<0.05). Conclusions After treated with glutamine and MSCs transplantation, the degree of intestinal ischemia reperfusion injury is obviously reduced in rats. It may be mediated through inhibiting the expression of caspase-3 and NF-kB and promoting the expression of Bcl-2.