Herein, corn silk extract and its flavonoids were used to inhibit the formation of Nε-carboxymethyllysine (CML) in a casein glucose-fatty acid model system. Under these optimum extraction conditions, nine major flavonoids were identified and quantified by HPLC-MS/MS. The percent inhibition of CML formation by corn silk extract was 76.57%. The inhibitory mechanism of corn silk extract toward CML formation was further investigated by examining the trapping of glyoxal/methyl glyoxal by the major flavonoids (5 mM) using HPLC-ESI-MS, and mono-, di-, and tri-adducts were found for some flavonoid compounds. The antioxidant activity of the corn silk extract was evaluated by the DPPH and ABTS assays. The scavenging activity of the corn silk extract for DPPH and ABTS was 84.38% and 89.11%, respectively. The results suggested that corn silk extract inhibited CML formation through glyoxal/methyl glyoxal scavenging or by its antioxidant activity attributed to its flavonoid content.