Inflammatory bowel disease (IBD) is a genetically pre‐determined, chronic, relapsing, inflammation of the intestinal epithelium. It is characterised by intestinal barrier dysfunction caused mainly by an alteration or redistribution of tight junction proteins that contributes towards increased permeability. Barrier disruption is also associated with enhanced activity of pro‐inflammatory cytokines such as TNF‐α, IFN‐γ and IL‐13. Although disease initiation in IBD remains elusive, inflammation results from an exaggerated immune response in genetically predisposed individuals, as well as altered responses to the luminal microbiota. In vivo studies suggest that pro‐inflammatory cytokines, such as TNF‐α and IFN‐γ, disrupt the intestinal barrier through effects on the tight junctions (TJ) and induction of apoptosis. This results in increased intestinal epithelial permeability that contributes to the pathogenesis of IBD. However, whether the effect of pro‐inflammatory cytokines on the TJs involves a loss or redistribution of TJ proteins is unknown. Here human colonic enteroids were used to investigate the effects of TNF‐α, IFN‐γ and a combination of TNF‐α and IFN‐γ on the expression and distribution of tight junction proteins, as they form the main paracellular barrier that determines intestinal epithelial permeability. Enteroids were grown from crypts isolated from transverse colonic biopsies from healthy patients, suspended in Matrigel© and overlaid with stem cell media. After 11d or 14d of growth, enteroids were exposed to TNF‐α (1, 10 or 100ng/mL), IFN‐γ (0.1, 1 or 10ng/mL) or a combination of TNF‐α/IFN‐γ (1/0.1, 10/1 or 100/10ng/mL), for either 24h or 72h. Quantification and localization of TJ proteins were determined by immunoblotting and immunofluorescent microscopy. Statistical significance was determined by a paired Student's t‐test. The lower concentrations of TNF‐α (1ng/mL and 10ng/mL), IFN‐γ (0.1ng/mL and 1ng/mL) and combination of TNF‐α/IFN‐γ (1/0.1 and 10/1ng/mL) had no effect on the TJs. In contrast, 24 h exposure to either 100 ng/mL TNF‐α, 10ng/mL IFN‐γ or 100 ng/ml TNF‐α plus 10ng/mL IFN‐γ caused a redistribution of the barrier forming TJ proteins, occludin and ZO‐1 and the pore‐forming tight junction protein, claudin‐2 from the junctions into the cytoplasm and the basolateral membrane of the epithelial cells, but did not affect total protein levels, whereas after 72h exposure to the same, there was a significant reduction in occludin and ZO‐1 (P<0.05) and a parallel increase in claudin‐2 (P<0.05). This was associated with a loss of occludin and ZO‐1 from the TJs, cytoplasm and the basolateral membrane of the epithelial cells. These data reflect a time‐dependent action of pro‐inflammatory cytokines on TJs of the human colonic epithelium, which involves an initial disruption of the barrier forming TJ proteins followed by an increase in the pore‐forming TJ protein. This action may result in increased epithelial colonic permeability in diseases such as IBD.Support or Funding InformationSupported by a University of Otago Research Grant and Grants from the Dean's Fund, Otago School of Medical Sciences and Dunedin School of Medicine and the Department of Physiology.