BACKGROUND AND PURPOSE: Inflammatory bowel disease (IBD) patients display genetic polymorphisms in toll-like receptor 4 (TLR4) genes, contributing to dysregulate enteric nervous system (ENS) circuits with increased levels of 5-HT and alteration of the neuroimmune crosstalk. In this study, we investigated the impact of TLR4 signalling on mouse ENS dysfunction caused by dextran sulphate sodium (DSS)-induced ileitis. EXPERIMENTAL APPROACH: Male C57BL/6J (wild-type [WT]) and TLR4(-/-) mice (10 +/- 2 weeks old) received 2% DSS in drinking water for 5 days and then were switched to 3-day regular drinking water. Histological analysis and proinflammatory cytokine mRNA levels were assessed in ileal samples. Gut motility was evaluated by changes in transit of a fluorescent-labelled marker and isometric neuromuscular responses of ileal full-thickness segments to receptor and non-receptor-mediated stimuli. Alterations in ENS architecture were assessed by confocal immunohistochemistry in longitudinal muscle-myenteric plexus whole-mount preparations. KEY RESULTS: In WT mice, DSS treatment caused delayed gastrointestinal transit, ileal myenteric neurodegeneration, reactive gliosis and release of proinflammatory cytokines. Enhanced cholinergic and tachykinergic excitatory tone, increased inducible nitric oxide synthase (iNOS)-mediated relaxation, and changes in 5-HT(2A) and 5-HT(3) receptor-mediated responses were observed during ileitis in WT mice. TLR4 deficiency reversed most of the functional and morphological abnormalities. CONCLUSION AND IMPLICATIONS: Our results demonstrate that TLR4 activity influences the severity of ileitis, neuroglial plasticity, gut motility, and nitrergic and 5-HTergic neurotransmissions. The neuroimmune interaction between TLR4 and 5-HT observed in our study appears to be a potential pharmacological target to treat ENS dysfunction implicated in IBD onset/progression.