Purpose: To investigate the inhibitory effect of the methanol fraction of Dioscorea nipponica Makino ethanol extract (DNM) on melanogenesis both in vitro and in vivo. Methods: Cultured mouse B16 melanoma cell and zebra fish were used to evaluate the melanogenesis inhibitory activity of DNM in vitro and in vivo, respectively. In B16 cells, inhibitory effects on intracellular melanogenesis, tyrosinase activity and reactive oxygen species (ROS) were determined after DNM treatment. In zebra fish, both toxic and anti-melanogenic activities of DNM on developed larvae were evaluated. Results: In B16 cells, the results show that DNM dose-dependently inhibited melanogenesis at non-toxic concentrations. Surprisingly, however, DNM had no effect on intracellular tyrosinase activity or the amount of the enzyme in B16 cells. On the other hand, DNM showed strong antioxidant activities against cell-free 2,2-diphenyl-1-picryl-hydrazl (DPPH) and 2,2'-azino-bis (3-etnylbenzthiazoline-6-sulphaonic acid) (ABTS(+)) free radical and intracellular ROS in B16 cells. In zebra fish, DNM significantly and dose-dependently inhibited skin melanogenesis of zebra fish larvae at non-toxic concentrations. Conclusion: The findings demonstrate that DNM inhibits melanogenesis in vitro in B16 melanoma cells and in vivo in zebrafish. Furthermore, DNM exhibits potent inhibition of melanogenesis probably as a result of its antioxidant activity in the cells.