Acetyl salicylic acid (ASA) is one of the most frequently prescribed medications for the secondary prevention of cardiovascular and cerebrovascular events. It has recently been reported to cause small intestinal mucosal injury at a considerably higher rate than previously believed. The aim of this study is to investigate the mechanism by which this occurs using an in vitro small intestine model focusing on the role of oxidative stress and cell permeability. Differentiated Caco-2 exhibits a phenotype similar to human small intestinal epithelium. We measured whether ASA induced the increase of differentiated Caco-2 permeability, the decrease of tight junction protein expression, the production of reactive oxygen species (ROS), and the expression of ROS-modified zonula occludens-1 (ZO-1) protein. In some experiments, Mn(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP, a superoxide dismutase mimetic) was used. The nontoxic concentration of ASA decreased transepithelial electrical resistance and increased the flux of fluorescein isothiocyanate-conjugated dextran across Caco-2 in a time-dependent manner. The same concentration of ASA significantly decreased ZO-1 expression among TJ proteins as assessed by Western blot and immunocytochemistry and increased ROS production and the expression of oxidative stress-modified ZO-1 protein. However, MnTMPyP suppressed the ASA-induced increased intercellular permeability and the ASA-induced ROS-modified ZO-1 expression. Our findings indicate that ASA-induced ROS production can specifically modify the expression of ZO-1 protein and induce increased cell permeability, which may ultimately cause small intestinal mucosal injury.