Abstract: AimTo evaluate the effect of paeonol on the activity of tyrosinase and provide experimental evidence for the treatment of hyperpigmentation disorders. MethodsTyrosinase activity was estimated by measuring the oxidation rate of L-3,4-dihydroxyphenylalanine (L-Dopa). The inhibitory effects of paeonol on the activity of mushroom tyrosinase and Michaelis-Menten kinetics were deduced from the Lineweaver-Burk plots. ResultsThe inhibitory concentration of paeonol leading to 50% enzyme activity lost (IC₅₀) was estimated to be 0.60 mmol·L^-1. The inhibition constants for paeonol binding free enzyme, K_I, and substrate-enzyme, K_IS, are 0.084 and 0.12 mmol·L^-1, respectively. Conclusion Paeonol is a potential mixed inhibitor of mushroom tyrosinase. The mixed inhibition function may originate from its ability to form a Schiff base with a primary amino group and to chelate copper at the active site of tyrosinase.