Tyrosinase (EC 126.96.36.199) is a key enzyme in post-harvest browning of fruit and melanin formation in human skin. Tyrosinase inhibitors are important in agricultural and cosmetic chemistry. In the present study, the inhibitory kinetics of enterolactone on themonophenolase and diphenolase activities of mushroom tyrosinasewere investigated using the kinetic method of substrate reaction. The results showed that enterolactone inhibited themonophenolase and diphenolase activities of mushroom tyrosinase, with IC50 values of 0.42 and 0.124 mM, respectively. The inhibition kinetics showed that enterolactone displayed non-competitive mechanism. The inhibition constants were determined to be 0.8315 and 0.6956mM, respectively. Furthermore, enterolactone exerted a potent inhibitory effect on intracellular melanin formation in B16/F10 murine melanoma cells and did not cause cytotoxicity. These results provide a comprehensive understanding of the inhibitory mechanisms underlying enterolactoneactivity and its inhibition of melanin formation, suggesting the potential benefits of using this compound.