Environmental factors such as diet are known to play important roles in inflammatory bowel disease (IBD). Epidemiological studies have indicated that a high-fat diet is a risk factor for IBD. In addition, the balance between effector T cells (T_eff) and regulatory T cells (T_reg) contributes to the pathogenesis of mucosal inflammation. The aim of this study was to understand the mechanisms by which a high-fat diet can regulate susceptibility to intestinal inflammation. Wild-type C57BL/6 mice were fed either a commercial high-fat diet or a normal diet, then exposed to dextran sulphate sodium (DSS) to induce colonic inflammation. Intraepithelial lymphocytes (IEL) were isolated from the colon, and their phenotype and cytokine profile were analysed by flow cytometry. Mice receiving the high-fat diet were more susceptible to DSS-induced colitis. They had higher numbers of non-CD1d-restricted natural killer (NK) T cells in the colonic IEL, when compared to mice fed a normal diet. These cells expressed tumour necrosis factor (TNF)-α and interferon (IFN)-γ, which are up-regulated by high-fat diets. Mice fed the high-fat diet also had decreased levels of colonic T_reg. Depletion of colonic NK T cells or adoptive transfer of T_reg reduced the DSS colitis in these mice, and reduced the colonic expression of TNF-α and IFN-γ. We conclude that a high-fat diet can increase non-CD1d-restricted NK T cells and decrease T_reg in the colonic IEL population. This altered colonic IEL population leads to increased susceptibility to DSS-induced colitis. This effect may help to explain how environmental factors can increase the susceptibility to IBD.