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. •• Rhein exerts an protective effect on intestinal barrier and has an anti-inflammagtory effect in vitro. • Rhein protects intestinal barrier from TNF-α stimulation by inhibiting NF-κB/MLCK/p-MLC activation. • Rhein reduces inflammatory mediators by inhibiting TLR4/NF-κB signals and NLRP3 inflammasome. [ABSTRACT FROM AUTHOR]