The ability of heartwood extract of Morus bombycis in controlling the melanogenesis in B16 melanoma was investigated. An inhibitory effect was observed when the dose concentration was less than 10 mg/L. When the dose was 10 mg/L, the extracellular melanin accumulated was only 5% of the control. Proteome analysis with 2-D PAGE and MALDI-TOF showed that there were various proteins involved in inhibitory melanogenesis. It seems that down-regulation of TRP2, vacuolar proton-translocating ATPase 100 kDa subunit isoform a1-I and V-ATPase E2 subunit were involved in melanosome development and regulation that could reduce melanin synthesis.