Increasing evidence has indicated the role the farnesoid X receptor (FXR) in maintaining metabolic homeostasis. In the ileum FXR promotes the transcription of fibroblast growth factor19 (FGF19), a hormone with positive effects on glucose metabolism, and expression of tight junction (TJ) proteins. Prior studies have indicated a link between type 2 diabetes (T2DM), reduced serum FGF19 levels and increased intestinal permeability, a well-known pathogenic factor of several cardio-metabolic disorders. However, it is currently unknown whether the exposure to high glucose (HG) may directly alter FXR/FGF19/TJ axis in human gut mucosa. To this end, we studied 60 subjects undergoing to colonoscopy with terminal ileum endoscopy and collection of ileal mucosa specimens. Subjects were classified on the basis of their glucose tolerance as having NGT, prediabetes and T2DM. Ileal levels of FXR, FGF19 and the TJ proteins Zonulin (ZO-1), occludin and claudin-1 were assessed by western blot and RT-PCR. Serum FGF19 concentrations were evaluated by ELISA assay. Ileal bioptic fragments obtained from subjects with NGT were cultured in presence of HG (25mM-50mM) or mannitol (M), as hyperosmolar control. As compared to NGT subjects, those with prediabetes and T2DM exhibited a reduction of intestinal FXR expression, which was paralleled by reduced ileal mRNA and serum levels of its downstream target FGF19. We also observed in subjects with prediabetes and T2DM an altered gut barrier function as demonstrated by lower TJ proteins expression associated with an up-regulation of the pro-inflammatory cytokines TNF-α, IL-1β and IL-6. Notably, HG exposure significantly reduced FXR expression levels and decreased FGF19 synthesis. Additionally, ileal mucosa biopsies incubated with HG exhibited a significant down-regulation of ZO-1, occludin and claudin-1 protein levels and higher expression of TNF-α, IL-1β and IL-6 as compared to the M-treated control. Our results demonstrate that individuals with altered glucose tolerance have a down-regulation of intestinal FXR/FGF19/TJ axis, and hyperglycemia may directly affect ileal FXR signaling and induce intestinal mucosa dysfunction.