The effects of Erigeron canadensis extract on melanogenesis and cell toxicity in cultured B16F10 mouse melanoma cells were investigated. E. canadensis extract down regulated melanin synthesis effectively at a non-toxic concentration. Its extract was fractionated by using a recycling HPLC with GS310 column (21.5×500 mm, 10-15 μM) into five fractions. The fraction 1 showed melanin inhibition by 48.0% at 100 mg/ml which was 2.5 times more efficient than the depigmenting effect of commercial arbutin (17.5%) and also did not show cell toxicity. To elucidate the depigmenting mechanism of fraction 1, in vitro and cellular tyrosinase activity, antioxidant activity, and protein level of the main melanogenic enzymes, such as tyrosinase, TRP-1 and TRP-2 were evaluated. Fraction 1 inhibited melanin synthesis in B16F10 melanoma cells by decreasing protein levels of melanogenic enzymes, especially tyrosinase. In conclusion, we suggest that this fraction may be a safe and effective depigmentation agent.