Dietary gliadin, which is a component of gluten, can induce alterations in intestinal microbiota and may cause severe disorders like celiac disease in gluten intolerant subjects. However, studies addressing the impact of gluten in gluten tolerant subjects are more limited. We hypothesized that subjects at risk for metabolic diseases may be more susceptible to adverse reactions to gliadin, and therefore chose to investigate the effect of gliadin in high-fat fed mice. This study is the first to show that gliadin as part of a defined synthetic high-fat feed alters intestinal microbiota, exacerbates glucose dysregulation and affects lipid accumulation in the liver as well as adipose tissue immune responses in wild type mice.