In this study, we investigated the detailed mode of action of 23R-AMA-induced inhibitory effects on cell proliferation and migration in B16-F10 melanoma, and found that these activities were caused by inhibitory regulation to both MITF expression and its transcriptional activity, and which were elicited by inhibition of β-catenin and c-Raf–MEK1–ERK signaling axis including FAK and c-Src. The gene discussed is MAP2K1; the disease is melanoma.