There is an increasing evidence that demonstrates a critical pathogenic role for the “gut-liver axis” in the development of nonalcoholic fatty liver diseases (NAFLD). Gut-derived endotoxin leakiness is increased under pathological conditions, including high fat diet (HFD) consumption. Gut fucosylation is a key force in maintaining a homeostatic relationship between the gut and its microbiota. Nevertheless, it is unclear how host fucosylation machinery contributes to obesity-associated intestinal barrier function, microbial translocation and inflammation. Glycosphingolipids (GSLs) are major constituents of enterocytes and consist of glycans conjugated to a lipid (ceramide) core. Ceramide is at the center of sphingolipid metabolism, and is hydrolyzed to generate sphingosine and fatty acid by ceramidases including neutral ceramidase (NcDase). However, whether gut NcDase contributes to intestinal fucosylation in NAFLD is unclear. We found that deletion of intestinal epithelial cells (IECs) NcDase induced marked upregulation of intestinal fucosylation in response to HFD exposure. Our preliminary studies further demonstrated that HFD-fed NcDase-/- mice have increases in intestinal AhR activity, IL-22 secretion and the production of fucosylated gangliosides (fucosyl-GM1). We also found that fucosyl-GM1 can bind to DC-SIGN, a C-type lectin, expressed in hepatic macrophages and can induce anti-inflammatory M2 macrophage. This has led to the central hypothesis that gut NcDase regulates the intestinal fucosylation and subsequent barrier function via IEC AhR signal; and that IECs NcDase-derived fucosylated glycosphingolipids induce hepatic macrophage polarization via fucosyl-GM1/DC-SIGN pathway and prevent the development of NAFLD. Importantly, the proposal also pursues translational studies that examine the efficacy of prebiotic 2′-FL feeding and fucosyl-GM1 treatment targeted at intestinal fucosylation and hepatic inflammatory macrophages in mitigating gut-liver axis changes. Following specific aims will be carried out: Aim 1: Determine the impact of NcDase on the gut fucosylation and the effects of this impact on intestinal barrier integrity. We will determine the impact of NcDase-related gut microbiota on the gut fucosylaton and barrier function. Aim 2: Determine whether NcDase regulation of IEC AhR contributes to gut fucosylation via microbial metabolite in response to HFD exposure. Aim3: Determine whether IEC NcDase-derived fucosylated gangliosides contribute to the induction of liver tolerogenic macrophages and evaluate the efficacy of fucosylation-based therapeutic interventions in NAFLD. Our studies on the NcDase-mediated gut fucosylation and prebiotic intervention will likely bring new mechanistic understanding and identify new therapeutic targets for the prevention and treatment of NAFLD.