The gut microbiota has an important role in the gut barrier, inflammation and metabolic functions. Studies have identified a close association between the intestinal barrier and metabolic diseases, including obesity and type 2 diabetes (T2D). Recently, Akkermansia muciniphila has been reported as a beneficial bacterium that reduces gut barrier disruption and insulin resistance. Here we evaluated the role of A. muciniphila-derived extracellular vesicles (AmEVs) in the regulation of gut permeability. We found that there are more AmEVs in the fecal samples of healthy controls compared with those of patients with T2D. In addition, AmEV administration enhanced tight junction function, reduced body weight gain and improved glucose tolerance in high-fat diet (HFD)-induced diabetic mice. To test the direct effect of AmEVs on human epithelial cells, cultured Caco-2 cells were treated with these vesicles. AmEVs decreased the gut permeability of lipopolysaccharide-treated Caco-2 cells, whereas Escherichia coli-derived EVs had no significant effect. Interestingly, the expression of occludin was increased by AmEV treatment. Overall, these results imply that AmEVs may act as a functional moiety for controlling gut permeability and that the regulation of intestinal barrier integrity can improve metabolic functions in HFD-fed mice.