Tiliroside was found to inhibit both monophenolase and diphenolase activity of mushroom tyrosinase. The lag time of tyrosine oxidation catalyzed by mushroom tyrosinase was obviously lengthened; 0.337 mM of tiliroside resulted in the lag time extension from 46.7 s to 435.1 s. A kinetic analysis shown that tiliroside was a competitive inhibitor for monophenolase and diphenolase with K(i) values of 0.052 mM and 0.26 mM, respectively. Furthermore, tiliroside showed 34.5% (p < 0.05) inhibition of intracellular tyrosinase activity and 54.1% (p < 0.05) inhibition of melanin production with low cytotoxicity on B16 mouse melanoma cells at 0.168 mM. In contrast, arbutin displayed 9.1% inhibition of cellular tyrosinase activity and 29.5% inhibition of melanin production at the same concentration. These results suggested that tiliroside was a potent tyrosinase inhibitor and might be used as a skin-whitening agent and pigmentation medicine.