To investigate the inhibitory effects of hydroxysafflor yellow A (HSYA) on the protein glycation in vitro. Using bovine serum albumin (BSA)-glucose assay, BSA-methylglyoxal (MGO) assay, and N-acetylglycyl-lysine methyl ester (G.K.) peptide-ribose assay, inhibitory effects of HSYA were investigated. Advanced glycation end products (AGEs) production was assessed by AGEs-specific fluorescence and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In BSA-glucose assay, HSYA concentration dependently decreased AGEs formation, with maximum inhibitory effects at 1 mM by 95%. Further more, HSYA also showed significant inhibitory effects on MGO-medicated protein modification and subsequent cross-linking of proteins. Finally, when co-incubated with G.K. peptide and ribose, HSYA exhibited its antiglycation effects, and the maximum inhibitory effects of HSYA at 1 mM were 84%. Overall, our present study provides the first evidence of the antiglycation effects of HSYA on AGEs formation in vitro.