Ischemic injury induces breakdown of the intestinal barrier. Recent studies in porcine post-ischemic tissues indicate that inhibition of NHE2 results in enhanced recovery of barrier function in vitro via a process involving interepithelial tight junctions. To further study this process, recovery of barrier function was assessed in wild type (NHE2^+/+) and NHE2^-/- mice in vivo and wild type mice in vitro. Mice were subjected to complete mesenteric ischemia in vivo, after which barrier function was measured by blood-to-lumen mannitol clearance over a 3-hour recovery period or measurement of transepithelial electrical resistance (TER) in Ussing chambers immediately following ischemia. Tissues were assessed for expression of select junctional proteins. Compared with NHE2^+/+ mice, NHE2^-/- mice had greater intestinal permeability during the post-ischemic recovery process. In contrast to prior porcine studies, pharmacological inhibition of NHE2 in post-ischemic tissues from wild type mice also resulted in significant reductions in TER. Mucosa from NHE2^-/- mice displayed a shift of occludin and claudin-1 expression to the Triton-X soluble membrane fractions and showed disruption of occludin and claudin-1 localization patterns following injury. This was qualitatively and quantitatively recovered in NHE2^+/+ mice as compared to NHE2^-/- mice by the end of the 3-hour recovery period. Serine phosphorylation of occludin and claudin-1 was down-regulated in NHE2^-/- post-ischemia compared with wild type mice. These data indicate an important role for NHE2 in recovery of barrier function in mice via a mechanism involving tight junctions.