Advanced glycation end products (AGEs) are dietary risk factors formed through the non-enzymatic glycation of reducing sugars with proteins, lipids, and other compounds. Siraitia grosvenorii polysaccharide (SGP) exhibits strong antioxidant activity and holds potential as a natural inhibitor of glycation. This study aims to investigate the anti-glycation activity and mechanisms of SGP, providing a theoretical basis for the anti-glycation effects of SGP. The results demonstrated that SGP inhibited the formation of AGEs during biscuit baking in a food matrix. In the bovine serum albumin-fructose (BSA-Fru) model, SGP reduced the formation of AGEs by chelating metal ions. SGP, Fru, and BSA were found to share the same binding sites, enabling SGP to compete with Fru for the aspartic acid 108 and arginine 144 binding sites on BSA, thereby directly inhibiting AGEs formation. In the Caco-2 cell model, SGP alleviated N-ε- (Carboxymethyl)-l-lysine (CML)-induced damage by reducing oxidative stress and regulating metabolic pathways, including the glycine-serine-threonine metabolism pathway, glyoxylate and dicarboxylate metabolism pathway, and the tricarboxylic acid (TCA) cycle. In summary, SGP not only serves as a natural inhibitor of in vitro AGEs formation but also alleviates intestinal barrier damage. This study provides a theoretical foundation for developing SGP as a functional food additive.