Intestinal anoxia/reoxygenation (A/R) injury induces loss of barrier function followed by epithelial repair. Myosin light chain kinase (MLCK) has been shown to alter barrier function via regulation of interepithelial tight junctions (TJ), but has not been studied in intestinal A/R injury. We hypothesized that A/R injury would disrupt TJ barrier function via MLCK activation and MLC phosphorylation. Caco-2BBe1 monolayers were subjected to anoxia for 2 hours followed by reoxygenation in 21% O₂, after which barrier function was determined by measuring transepithelial resistance (TER) and FITC-dextran flux. TJ proteins and MLCK signaling were assessed by western blotting, real time-PCR, or immunofluorescence microscopy. The role of MLCK was further investigated with select inhibitors (ML-7 and peptide 18) using in vitro and ex vivo models. Following A/R injury, there was a significant increase in paracellular permeability compared to control cells, as determined by TER and dextran fluxes (P<0.05). The TJ protein occludin was internalized during A/R injury and re-localized to the region of the TJ after 4h recovery. MLC phosphorylation was significantly increased by A/R injury (P<0.05) and treatment with the MLCK inhibitor peptide 18 attenuated the increased epithelial monolayer permeability and occludin endocytosis caused by A/R injury. Application of MLCK inhibitors to ischemia-injured porcine ileal mucosa induced significant increases in TER and reduced mucosal-to-serosal fluxes of 3H-labeled mannitol. These data suggest that MLCK-induced occludin endocytosis mediates intestinal epithelial barrier dysfunction during A/R injury. Our results also indicate that MLCK-dependent occludin regulation may be a target for the therapeutic treatment of ischemia/reperfusion injury.