This study was conducted to evaluate the effects of Glycyrrhizae Radix water extract, known as depigmenting agent, on melanin biosynthesis in the HM3KO human melanoma cells. The inhibitory effect of Glycyrrhizae Radix water extract on melanogenesis was identified by mushroom tyrosinase assay in vitro. To determine whether Glycyrrhizae Radix water extract suppress melanin synthesis in cellular level, HM3KO cells were cultured in the presence of different concentrations of Glycyrrhizae Radix water extract and the effects on cell proliferation, melanin contents and tyrosinase activity were examined after 3 days. Treatment with Glycyrrhizae Radix at various concentrations did not exhibit any change of cell viability, and increased the cell proliferation. And the water extract of Glycyrrhizae Radix inhibited melanin contents and tyrosinase activity in a dose-dependent manner, compared with untreated group. These results suggest that the inhibitory effect of Glycyrrhizae Radix water extract on melanogenesis is due to the suppression of tyrosinase in HM3KO cells.