Matrix metalloproteinase-9 (MMP-9) has been implicated as being an important pathogenic factor in inflammatory bowel disease (IBD). MMP-9 is markedly elevated in intestinal tissue of patients with IBD, and IBD patients have a defective intestinal tight-junction (TJ) barrier manifested by an increase in intestinal permeability. The loss of intestinal epithelial barrier function is an important contributing factor in the development and prolongation of intestinal inflammation; however, the role of MMP-9 in intestinal barrier function remains unclear. The purpose of this study was to investigate the effect of MMP-9 on the intestinal epithelial TJ barrier and to delineate the intracellular mechanisms involved by using in vitro (filter-grown Caco-2 monolayers) and in vivo (mouse small intestine recycling perfusion) systems. MMP-9 caused a time- and dose-dependent increase in Caco-2 TJ permeability. MMP-9 also caused an increase in myosin light-chain kinase (MLCK) gene activity, protein expression, and enzymatic activity. The pharmacological MLCK inhibition and siRNA-induced knockdown of MLCK inhibited the MMP-9-induced increase in Caco-2 TJ permeability. MMP-9 caused a rapid activation of the p38 kinase signaling pathway and inhibition of p38 kinase activity prevented the MMP-9-induced increase in MLCK gene activity and the increase in Caco-2 TJ permeability. MMP-9 also caused an increase in mouse intestinal permeability in vivo, which was accompanied by an increase in MLCK expression. The MMP-9-induced increase in mouse intestinal permeability was inhibited in MLCK-deficient mice. These data show for the first time that the MMP-9-induced increase in intestinal TJ permeability in vitro and in vivo was mediated by the p38 kinase signal transduction pathway upregulation of MLCK gene activity and that therapeutic targeting of these pathways can prevent the MMP-9-induced increase in intestinal TJ permeability. NEW & NOTEWORTHY MMP-9 is highly elevated in patients with IBD. IBD patients have compromised intestinal TJ barrier function manifested by an increase in intestinal permeability and intestinal inflammation. This study shows that MMP-9, at clinically achievable concentrations, causes an increase in intestinal TJ permeability in vitro and in vivo. In addition, a MMP-9-induced increase in intestinal TJ permeability was mediated by an increase in MLCK gene and protein expression via the p38 kinase pathway.