Advanced glycation end-products (AGEs) generated during protein glycation trigger significant concerns for human health. Exploring safe and effective natural ingredients exhibiting excellent AGEs inhibition has become a promising way to overcome this issue. This research focused on the structural features of Diaphragma Juglandis Fructus polysaccharide (FJP-1) as well as its AGE-inhibiting capacity and mechanisms. The results demonstrated that FJP-1 with a Mw of 22.54 kDa was composed of galactose (Gal), rhamnose (Rha), glucuronic acid (Glc-UA), galacturonic acid (Gal-UA), glucose (Glc) and arabinose (Ara) at a molar ratio of 9.58:3.52:2.72:74.95:1.52:7.71. Its backbone was probably composed of → 4)-Galp-UA-(1→, with branches of → 4,6)-Galp-(1 → and → 4)-Glcp-(1→. And the terminal branches were T-Rhap-(1→), T-Galp-(1→) and T-Araf. In three in vitro models of anti-glycosylation, FJP-1 effectively inhibited the formation of total and special fluorescent AGEs, reduced fructosamine levels, and prevented structural modifications, oxidation, and cross-linking of the protein. Mechanistic and multispectral assays elucidated that the anti-glycative mechanisms of FJP-1 were achieved by capturing methylglyoxal, scavenging free radicals, and binding to bovine serum albumin to form adducts. Pearson's correlation analyses showed that there were positive correlations between FJP-1's antioxidant property with the AGEs formation. This study demonstrated that FJP-1 is a potential AGEs inhibitor for development of functional foods and treatment various diseases mediated by AGEs and oxidative stress.