Mycophenolic acid (MPA) may cause gastrointestinal adverse effects by damaging the intestinal epithelial barrier, the underlying mechanisms remain elusive. Studies have demonstrated that oxidative stress caused by reactive oxygen species (ROS) is linked to tight junction (TJ) proteins and apoptosis, both of which cause abnormalities in intestinal barrier function. Mitochondria, one of the main sources of ROS and abnormally high levels of ROS are linked to mitochondrial dysfunction. The aim of this study was to investigate whether MPA induces intestinal barrier dysfunction through regulation of the mitochondrial ROS. MPA-induced intestinal injury model in Kunming mice and Caco-2 cells. The effect of MPA on Caco-2 cell viability was measured by MTT; tissue diamine oxidase and endotoxin expression were determined by ELISA; expression of total proteins of ZO-1, occludin, Bax, Bcl-2, and mitochondrial proteins of Cytochrome C and Bax was measured by Western blot; and the localization of Cytochrome C with MitoTraker was observed by immunofluorescence staining. Caco-2 cell apoptosis, ROS levels, and mitochondrial membrane potential were detected by flow cytometry, while intramitochondrial ROS levels were observed by MitoSOX fluorescence staining. The results showed that MPA increased intracellular and mitochondrial ROS production to promote oxidative stress and the antioxidant NAC effectively restored ZO-1 and occludin expressions, reduced apoptosis in intestinal epithelial cells. Furthermore, we found that low concentrations of MPA caused mitochondrial damage, induced hyperpolarization of the mitochondrial membrane potential and the translocation of Cytochrome C and Bax proteins from the cytoplasm to the mitochondria. The mitochondrial protectant SS-31 reduces intracellular and intramitochondrial ROS, upregulates TJ, and reduces apoptosis. Our studies suggest that MPA-induced intestinal barrier dysfunctionandis mediated, at least in part, by impairing mitochondrial function and promoting oxidative stress.