Reactive oxygen species (ROS) generated by administration of lipopolysaccharide (LPS) are known to cause intestinal barrier dysfunction. When ROS production was quantified with L-012 by in vivo imaging system, the bioluminescent signal of L-012 was markedly increased in the abdomen of mice treated with LPS (6 mg/kg i.p.). However, increased ROS production was significantly attenuated in Nox1- or iNOS-deficient mice. In wild-type mice (WT), administration of LPS elicited a marked increase in intestinal mucosal permeability determined by the amount of fluorescein isothiocyanate-labeled dextran (FD-4) transferred from the gut lumen into circulation. Increased intestinal permeability induced by LPS was significantly suppressed in Nox1- or iNOS-deficient mice. While histological analysis demonstrated no apparent change in the intestinal structure, decreased levels of tight junction proteins, occludin and ZO-1, were observed in the ileum of WT treated with LPS. At the same time, activation of matrix metalloproteinase-9 (MMP9) was depicted in the ileum of WT but not of Nox1-deficient mice. Taken together, ROS derived from NOX1 may increase the activity of MMP9 to elicit intestinal barrier dysfunction during endotoxemia.