Defects in the gut epithelial barrier have now been recognized to be responsible for diabetic endotoxemia. In everyday life, Mulberry leaf tea is widely used in Asian nations due to its proposed benefits to health and control of diabetes. Evidence indicates the potential role of Kuwanon G (KWG), a component from Morus alba L., on blocking the gut epithelial barrier. In lipopolysaccharides (LPS)-damaged Caco-2 cells, it was found that KWG increased the viability of cells in a concentration-dependent manner. KWG administration significantly elevated the anti-oxidant abilities via increasing ratio of superoxidase dismutase (SOD)/malondialdehyde (MDA) and decreasing reactive oxygen species (ROS) within the cells. During KWG incubation, pro-inflammatory cytokines including interleukin (IL)-1β and tumor necrosis factor (TNF)-α were significantly reduced, tight junction proteins including zonula occludens (ZO)-1, intercellular adhesion molecule (ICAM)-1 and Occludin were dramatically increased as detected by immunofluorescence assay, trans-epithelial electrical resistance was significantly increased and the transmission of albumin-fluorescein isothiocyanate (FITC) across the barrier was decreased. In conclusion, the present study demonstrated that KWG could ameliorate LPS-induced disruption of the gut epithelial barrier by increasing cell viability and tight junction between cells, and decreasing pro-inflammatory cytokines and oxidative damage.