BACKGROUND: Methylglyoxal (MGO), a highly reactive precursor of advanced glycation end products (AGEs), is endogenously produced and prevalent in various ultra-processed foods. MGO has emerged as a significant precursor implicated in the pathogenesis of type 2 diabetes and neurodegenerative diseases. To date, the effects of dietary MGO on the intestine have been limited explored. Thus, this study investigates the impact of prolonged oral administration of MGOs on gut health in aged mice. METHODS: Aged mice received MGO chronically (100 mg/kg/day) for 4 weeks Intestinal samples were analyzed using RT-PCR and immunohistochemistry for proinflammatory cytokines, permeability markers, and tight junction proteins. 16S rRNA gene-based microbiome analysis was also performed to characterize microbiome composition and its metabolic potential. RESULTS: MGO treatment induced notable alterations at the intestinal level, characterized by an increased formation of MGO-glycated proteins with a concurrent induction of a pro-inflammatory status and reduced expression and delocalization of zonulin-1 and occludin, tight junction proteins. Changes in intestinal morphology were also observed, including hyperproliferation of Paneth cells and an augmented thickness of the intestinal mucus layer, as indicated by immunohistochemical data from MGO-treated mice. Investigation into the microbiota composition revealed that MGO is effective in selectively modifying its composition and metabolic pathways. A decreased abundance of bacterial genera associated with the production of acetic and butyric acids (i.e. Harryflintia, Intestinimonas and Ruminococcaceae genera) and a substantial increase in Lachnospiraceae and Akkermansia genera were found in MGO-treated mice. CONCLUSION: These findings highlight how dietary MGO can affect intestinal balance, providing valuable insights into the potential links between glycotoxins, gut microbiota, and overall gut functionality.