Berberine is one of the main alkaloids of Rhizoma coptidis which has been used for patients with gastrointestinal disorders. The major aim of this study was to investigate the effect of berberine on tight junction. Caco-2 cells were treated with various concentration of berberine. We observed the integrity of tight junction by measuring the transepithelial electrical resistance (TEER), and also studied the effect of berberine on morphology of tight junction and tight junction protein. These findings showed the first time that berberine could reduce epithelial gut permeability, and might help explain the possible mechanisms of anti-diarrhea activity of berberine.