Inflammatory bowel diseases (IBDs) are chronic, inflammatory disorders of the gastrointestinal tract with unclear aetiologies. Intestinal epithelial cells (IECs), containing crypt and villus enterocytes, occupy a critical position in the pathogenesis of IBDs and are a major producer of immunoregulatory cytokines and a key component of the intact epithelial barrier. Previously, we have reported that miR-200b is involved in the progression of IBDs and might maintain the integrity of the intestinal epithelial barrier via reducing the loss of enterocytes. In this study, we further investigated the impact of miR-200b on intestinal epithelial inflammation and tight junctions in two distinct differentiated states of Caco-2 cells after TNFα treatment. We demonstrated that TNFα-enhanced IL-8 expression was decreased by miR-200b in undifferentiated IECs. Simultaneously, miR-200b could alleviated TNFα-induced tight junction (TJ) disruption in well-differentiated IECs by reducing the reduction in the TEER, inhibiting the increase in paracellular permeability and preventing the morphological redistribution of TJ proteins Claudin1 and ZO-1. The expression levels of the JNK/C-JUN/AP-1 and MLCK/P-MLC pathways were attenuated in undifferentiated and differentiated enterocytes, respectively. Furthermore, a dual-luciferase reporter gene detection system provided direct evidence that C-JUN and MLCK were the specific targets of miR-200b. Collectively, our results highlighted that miR-200b played a positive role on IECs via suppressing intestinal epithelial IL-8 secretion and attenuating TJ damage in vitro, which suggested that miR-200b might be a promising strategy for IBD therapy.