Since ancient times, honeybee (Apis mellifera L.) venom (BV) has been applied to the skin as an anti-ageing remedy. To further access BV as an effective whitening agent for cosmetics and potential external treatment for topical use, we investigated its ability to inhibit tyrosinase activity and subsequent melanin biosynthesis. B16F1 melanoma cells were treated with 10 nM α-melanocyte stimulating hormone (α-MSH) and then with various doses of BV. BV inhibited direct tyrosinase activity and cellular tyrosinase activity, which decreased melanin synthesis in α-MSH-stimulated B16F1 melanoma cells. In addition, we used reverse transcription-polymerase chain reaction and Western blotting for melanogenesis-related genes such as tyrosinase-related protein (TRP)-1 and TRP-2 to examine the mechanisms underlying the inhibitory effects of BV on melanogensis. BV decreased the mRNA and protein expression of TRP-1 and TRP-2. These findings suggest that BV induced the downregulation of melanogenesis by inhibiting TRP-1 and TRP-2 activation.