Advanced glycation end products (AGEs) are prevalent in thermally processed foods. Long-term intake of AGEs has been linked to intestinal barrier damage, contributing to the development of chronic diseases. Galacto-oligosaccharides (GOS) restored mucus barrier damage induced by dietary AGEs, but the underlying mechanisms remained unclear. This study focused on investigating the effects of GOS on mucin synthesis-related glycosyltransferases, as well as differentiation and apoptosis of goblet cells (GCs) in dietary AGEs-fed C57BL/6 mice. Fecal microbiota transplantation (FMT) was used to probe a possible contribution of the intestinal microbiota. The findings demonstrated that GOS upregulated the mRNA expression of glycosyltransferases (St3gal1 and St3gal3) while inhibiting p-ERK signaling. In addition, no marked changes in mRNA expression of the Wnt, BMP, Notch, and CHOP pathways (which are involved in GC proliferation, differentiation, and apoptosis) were observed following GOS intervention. FMT data suggested the hypothesis that the gut microbiota may participate in mediating changes in St3gal1 and p-ERK/ERK in response to GOS. These findings provided valuable insights into potential interventions for intestinal mucus barrier damage induced by AGEs.