The aim of this study was to decipher the mechanisms by which the major human milk oligosaccharide (HMO), 2’-fucosyllactose (2’FL), can affect body weight and fat mass gain upon high-fat diet (HFD) feeding in mice. In particular, we wanted to elucidate whether 2’FL metabolic effects are linked with changes in intestinal mucus production and secretion, mucin glycosylation and degradation, as well as with the modulation of the gut microbiota, fecal proteome and endocannabinoid (eCB) system.