Recent studies have implicated a pathogenic role for matrix metalloproteinases 9 (MMP-9) in inflammatory bowel disease. Although loss of epithelial barrier function has been shown to be a key pathogenic factor for the development of intestinal inflammation, the role of MMP-9 in intestinal barrier function remains unclear. The aim of this study was to investigate the role of MMP-9 in intestinal barrier function and intestinal inflammation. Wild-type (WT) and MMP-9^−/− mice were subjected to experimental dextran sodium sulfate (DSS) colitis by administration of 3% DSS in drinking water for 7 days. The mouse colonic permeability was measured in vivo by recycling perfusion of the entire colon using fluorescently labeled dextran. The DSS-induced increase in the colonic permeability was accompanied by an increase in intestinal epithelial cell MMP-9 expression in WT mice. The DSS-induced increase in intestinal permeability and the severity of DSS colitis was found to be attenuated in MMP-9^−/− mice. The colonic protein expression of myosin light chain kinase (MLCK) and phospho-MLC was found to be significantly increased after DSS administration in WT mice but not in MMP-9^−/− mice. The DSS-induced increase in colonic permeability and colonic inflammation was attenuated in MLCK^−/− mice and MLCK inhibitor ML-7-treated WT mice. The DSS-induced increase in colonic surface epithelial cell MLCK mRNA was abolished in MMP-9^−/− mice. Lastly, increased MMP-9 protein expression was detected within the colonic surface epithelial cells in ulcerative colitis cases. These data suggest a role of MMP-9 in modulation of colonic epithelial permeability and inflammation via MLCK.