Barrier defects and/or alterations in the ability of the gut epithelium to repair itself are critical etiological mechanisms of gastrointestinal disease. Our ongoing studies indicate that the chemokine receptor CXCR4 and its cognate ligand CXCL12 regulate intestinal-epithelial barrier maturation and restitution in cell culture models. Gene-deficient mice lacking CXCR4 expression specifically by the cells of the intestinal epithelium were used to test the hypothesis that CXCR4 regulates mucosal barrier integrity in vivo. Epithelial expression of CXCR4 was assessed by RT-PCR, Southern blot, immunoblot and immunohistochemistry. In vivo wounding assays were performed by addition of 3% dextran sodium sulfate (DSS) in drinking water for 5 days. Intestinal damage and DAI scores were assessed by histological examination. Extracellular-regulated kinase (ERK) phosphorylation was assessed in vivo by immunoblot and immunofluorescence. CXCR4 knockdown cells were established using a lentiviral approach and ERK phosphorylation was assessed. Consistent with targeted roles in restitution, epithelium from patients with inflammatory bowel disease indicated that CXCR4 and CXCL12 expression was stable throughout the human colonic epithelium. Conditional CXCR4-deficient mice developed normally, with little phenotypic differences in epithelial morphology, proliferation or migration. Re-epithelialization was absent in CXCR4 conditional knockout mice following acute DSS-induced inflammation. In contrast, heterozygous CXCR4-depleted mice displayed significant improvement in epithelial ulcer healing in acute and chronic inflammation. Mucosal injury repair was correlated with ERK1/2 activity and localization along the crypt-villus axis, with heterozygous mice characterized by increased ERK1/2 activation. Lentiviral depletion of CXCR4 in IEC-6 cells similarly altered ERK1/2 activity and prevented chemokine-stimulated migration. Taken together, these data indicate that chemokine receptors participate in epithelial barrier responses through coordination of the ERK1/2 signaling pathway.