Methylglyoxal (MGO), a cytotoxic factor, reacts irreversibly with the side chains of lysine, cysteine, and arginine residues in proteins to form advanced glycation end products (AGEs)which might be a major pathological factor associated with diabetic complications. Thus, it is necessary to prevent or alleviate such diseases through inhibiting the formation of AGEs or lowering these AGEs-induced cellular damages. Based on our previous work, it was known that phloretin, an apple polyphenol, can inhibit the formation of AGEs under simulated physiological conditions. In this study, we found that phloretin prevented the formation of AGEs through trapping MGO in human umbilical endothelial cells (HUVECs). The phloretin-MGO adducts were analyzed in PBS and HUVECs. Surprisingly, only 1 MGO-phloretin adduct was detected in HUVECs, which was formed within 0.5 h and metabolized eventually within 24 h. The specific phloretin-MGO adduct was synthesized and identified by MS and NMR analysis. Its anti-inflammatory effect against AGEs was further investigated together with the parent compound, phloretin, which was proved to be through RAGE/p38 MAPK/NF-κB signaling pathway. Taken together, our data indicated the positive role of phloretin-MGO adduct on phloretin's protective effects, which might offer a new insight into the action mechanism of polyphenols against AGEs-induced damages.