Celiac disease (CD) is a gluten intolerance caused by a T cell response against HLA-DQ2 and DQ8-bound gluten peptides. Some subjects experience gastrointestinal symptoms in the absence of villous atrophy. Here we investigate the potential mechanisms of gut dysfunction in gluten sensitive HLA-DQ8 transgenic mice. HLA-DQ8 mice were sensitized and gavaged with gliadin 3xweek, for 3 weeks (G/G). Controls included a) non-sensitized mice gavaged with rice (C), b) gliadin-sensitized mice gavaged with rice (G/R), c) BSA-sensitized mice gavaged with BSA (BSA/BSA). CD3+ intra-epithelial lymphocyte (IEL), macrophage and FOX-P3 positive cell counts were determined. Acetylcholine (ACh) release, small intestinal contractility, and epithelial ion transport (Isc) were measured. Gut function was investigated after gluten withdrawal and in HLA-DQ6 mice. Intestinal atrophy was not observed in G/G mice. Recruitment of IELs, macrophages and FOX-P3+ cells were observed in G/G, but not in C, G/R or BSA/BSA mice. This was paralelled by increased ACh release from the myenteric plexus, muscle hypercontractility, and increased active ion transport in G/G mice. Changes in in muscle contractility normalized in DQ8 mice after a gluten withdrawal. HLA-DQ6 controls did not exhibit the abnormalities in gut function observed in DQ8 mice. Gluten sensitivity in HLA-DQ8 mice induces immune activation in the absence of intestinal atrophy. This is associated with cholinergic dysfunction and a pro-secretory state that may lead to altered water movements and dysmotility. The results provide a mechanism by which gluten could induce gut dysfunction in patients with a genetic predisposition but without fully evolved CD.