Objective To study the effects of Eclipta prostrata L (Mohanlian) ethanol extract, which activates tyrosinase in vitro, on melanogenesis and expression of its related genes(TYR, TRP-1/2 mRNA) in cultured B16 melanoma cells. Methods Using brownish guinea pigs as experimental models, cells containing melanin granules and DOPA-positive cells in 100 basal cells were counted by Schmorl and DOPA-oxidase stain methods respectively. The cells proliferation, melanogenesis and tyrosinase activity in cultured B16 cells were measured by MTT method, NaOH method and oxidative DOPA reaction respectively. The expression of TYR and TRP-1/2 mRNA were measured by RT-PCR. Results The ethanol extract of Eclipta prostrata L increased the cells with melanin granules in epidermal basal layer of guinea pigs and the DOPA-positive cells in epidermis. It also promoted melanogenesis and tyosinase activity(P<0.05) in B16 melanoma cells, and up-regulated TYR mRNA , but had no effects on TRP-1/2 mRNA. Conclusion Eclipta prostrata L ethanol extract have effects on activating melanogenesis and up-regulating TYR mRNA, which may be useful in treating vitiligo and promising in developing new drugs for treatment of vitiligo.