Mangosteen (Garcinia mangostana L) fruit contains many xanthones in its pericarp, such as α-mangostin. Here, we aimed to elucidate the physiological effect of α-mangostin and the mechanism on melanogenesis in mouse B16F10 cells. The melanin production in B16F10 cells was decreased by α-mangostin treatment. α-Mangostin also suppressed the enzymatic activity of tyrosinase, the critical enzyme for melanin synthesis. Furthermore, Western blot analysis revealed that α-mangostin down-regulated the protein quantity of tyrosinase, tyrosinase relative protein (TRP)-2, and microphthalmia-associated transcription factor (MITF). We also used inhibitors of the extracellular signal-regulated kinase (ERK), and glycogen synthase kinase 3 (GSK-3β) to identify the upstream signaling cascade of MITF. Results showed us GSK3β plays a more important role in α-mangostin regulated melanogenesis. Further, the de-pigmentation effect on normal human epidermal melanocytes (NHEMs) of α-mangostin was also confirmed. These results suggested that α-mangostin is a reagent for depigmentation and it has the potential to be applied as a component of cosmetics or pharmaceuticals for the therapy of spots, chloasma, or melanosis.