Objective To explore the impact and mechanism of miR-200b on intestinal epithelial tight junction. Methods The negative-lentivirus and human-miR-200b-lentivirus were employed to infect the Caco-2 cell thus establishing two stable cell lines which were then stimulated by 10 ng/mL human tumor necrosis factor-α (TNF-α) to establish the model of the intestinal epithelial injury. Those Caco-2 cells were divided into NC, NC+TNF-α, 200b, and 200b+TNF-α groups.The tight junction permeability was detected by transepithelial electrical resistance (TEER) and Fluorescein isothiocyanate-labeled dextran (FITCdextran). The protein alterations myosin light chain kinase (MLCK)/phosphorylated myosin light chain (P-MLC) pathways were measured by Western blot analysis. Results Compared to NC group, NC+TNF-α group had lower TEER, higher FITC-dextran, and up-regulated expressions of MLCK and P-MLC proteins (P < 0.05). Compared to NC+TNF-α group, 200b+TNF-α group had higher TEER, lower FITC-dextran and down-regulated expressions of MLCK and P-MLC proteins (P<0.05). Conclusion miR-200b ameliorated TNF-α-induced intestinal epithelial tight junction disruption via regulation MLCK/P-MLC pathway.