We previously reported that hederacolchiside A1 (Hcol A1), a new oleanene saponin isolated from Hedera colchica Koch (Araliaceae) exhibits a preferential cytotoxicity on a pigmented melanoma cell line. The present study confirms the high susceptibility of melanoma cell lines to this drug and shows concentrations producing a 50% decrease in cell content (IC50 values) inversely proportional to the melanin content of each cell line. At cytotoxic concentrations, Hcol A1 induces membrane-damaging effects within 6 h, cytoplasmic vacuolization within 24 h, and non-apoptotic cell death within 48 h. Using a new high-resolution magic-angle spinning nuclear magnetic resonance method, we have shown for the first time that this hederasaponin specifically interacts with melanin. The dissociation constant (2.7 mM) is comparable to those observed with drugs known to interact with melanin. Taking into consideration that the IC50 values were inversely proportional to the melanin in each cell line, our data suggest that, in addition to the delayed membrane injury induced by this drug, the ability of Hcol A1 to bind melanin could contribute to the higher toxicity of Hcol A1 in pigmented melanoma cells.