Activation of Aryl hydrocarbon receptor (AhR) is involved in the control of intestinal mucosal homeostasis. Intestinal barrier dysfunction contributes to the development of many intestinal diseases, such as inflammatory bowel disease (IBD). In this study, we investigated the mechanisms of AhR activation in the maintenance of intestinal barrier function. Adult C57BL/6 mice were treated with dextran sulphate sodium (DSS) for 7 days, with or without 6-Formylindolo(3,2-b)carbazole (FICZ), a ligand of AhR. We found that AhR activation by FICZ attenuated the decreased TJ protein expression in the colonic mucosa of the DSS-induced mice. Further, the increase of both MLC phosphorylation and MLCK expression in the mice with DSS-induced colitis was also significantly inhibited by FICZ induced AhR activation. For in vitro experiments, Caco-2 cells were treated with tumour necrosis factor alpha (TNF-α)/interferon gamma (IFN-γ) for 48 h, with or without FICZ. AhR activation prevented TNF-α/IFN-γ-induced decrease in TER and morphological disruption of the TJs in Caco-2 monolayers. It also inhibited TNF-α/IFN-γ-induced increase in MLCK expression and MLC phosphorylation by suppression of NF-κB p65 signaling pathway. Thus, AhR-activating factors might have potential as therapeutic agents for the treatment of patients with IBD.