Although enteric glial cells (EGCs) have been demonstrated to play a key role in maintaining intestinal epithelial barrier integrity, it is not known how EGCs regulate this integrity. We therefore hypothesized that glial‐derived neurotrophic factor (GDNF) produced by EGCs might be involved in this regulation. Here we investigated the role of GDNF in regulating epithelial barrier function in vivo. Recombinant adenoviral vectors encoding GDNF (Ad‐GDNF) were administered intracolonically in experimental colitis induced by dextran sulphate sodium (DSS). The disease activity index (DAI) and histological score were measured. Epithelial permeability was assayed using Evans blue dye. The anti‐apoptotic potency of GDNF in vivo was evaluated. The expression of tumour necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), and myeloperoxidase (MPO) activity were measured by ELISA assay and/or RT‐PCR. The expression of ZO‐1, Akt, caspase‐3, and NF‐κB p65 was analysed by western blot assay. Our results showed that GDNF resulted in a significant reduction in enhanced permeability, inhibited MPO activity, IL‐1β and TNF‐α expression, and increased ZO‐1 and Akt expression. Moreover, GDNF strongly prevented apoptosis in vivo and significantly ameliorated experimental colitis. Our findings indicate that GDNF participates directly in restoring epithelial barrier function in vivo via reduction of increased epithelial permeability and inhibition of mucosal inflammatory response, and is efficacious in DSS‐induced colitis. These findings support the notion that EGCs are able to regulate intestinal epithelial barrier integrity indirectly via their release of GDNF in vivo. GDNF is namely an important mediator of the cross‐talk between EGCs and mucosal epithelial cells. GDNF may be a useful therapeutic approach to the treatment of inflammatory bowel disease. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.