In this study, the processes of differentiation and melanogenesis induced by 5,7-dimethoxycoumarin in murine (B16) and human (A375) melanoma cells were investigated. Taking into account the previously demonstrated antiproliferative and differentiation activities of this compound, we examined Ras/Raf/Mek/Erk mitogen-activated protein kinase activity following treatment; inhibition of Mek 1/2 kinase activity and subsequent reduction in Erk 1/2 activation were detected in both cell types. We observed melanogenesis induction associated to an increase in cAMP-response element-binding protein (CREB) and microphthalmia-associated transcription factor (Mitf) expression, both involved in its regulation. Mitf is fundamental for development, survival and differentiation of melanocyte and melanoma, since it regulates transcription of genes encoding for proteins involved in cell cycle progression or in melanogenesis, such as the enzyme tyrosinase. A significant increase of tyrosinase activity was revealed following treatment in B16 but not in A375 cells, although a strong synthesis of melanin was induced by 5,7-dimethoxycoumarin in both cell lines. The treatment induced protoporphyrine IX accumulation involved in melanogenesis since it promotes stability of cAMP. Finally, the Mek 1/2 inhibitor U0126 significantly potentiated growth inhibition of B16 cells triggered by 5,7-dimethoxycoumarin, suggesting that down-regulation of Raf/Mek/Erk pathway sensitizes melanoma cells to 5,7-dimethoxycoumarin treatment.