Intestinal epithelial barrier and intestinal inflammation play indispensable roles in the development of intestinal diseases. The major aims of the current study were to investigate the potential of rhein, a major flavonoid compound isolated from Rheum rhabarbarum, in the treatment of intestinal diseases and its underlying mechanisms in vitro.The protective role of rhein on intestinal epithelial barrier was evaluated in a monolayer of IEC-6 cells stimulated by TNF-α, while the anti-inflammatory effects were investigated in an IEC-6 cell model with LPS stimulation.Rhein inhibited the increase of phenol red flux and the decrease of TEER, as well as recovered the expression and distribution of ZO-1 and weakened MLC phosphorylation, MLCK expression and NF-κB activation. Meanwhile, LPS-stimulated IL-1β and IL-6 were down-regulated, expression levels of TLR4, NLRP3 and cleaved caspase1 were weakened and NF-κB was inactivated.These results suggested that rhein has potential therapeutic effects against intestinal diseases by maintaining intestinal epithelial barrier and suppressing intestinal inflammation.