Arbutin, a natural polyphenol isolated from the bearberry plant Arctostaphylos uvaursi, possesses whitening and anticancer properties. The effects of arbutin on melanogenesis and its pro-apoptotic effect on B16 murine melanoma cells have not yet been reported. In the present study, acetylated arbutin was prepared in order to improve the biological effects of arbutin, and it was found to significantly inhibit the biosynthesis of melanin and tyrosinase activity compared with parent arbutin in B16 murine melanoma cells. Interestingly, only acetylated arbutin strongly inhibited B16 murine melanoma cell migration in a dose-dependent manner. Both arbutin and acetylated arbutin significantly reduced cell viability, promoted cell apoptosis, caused G1 cell cycle arrest and induced mitochondrial disruption in B16 murine melanoma cells. Furthermore, reduced expression of B-cell lymphoma‑extra large (Bcl-xL) and Bcl-2 were observed in arbutin- and acetylated arbutin-treated cells. Therefore, arbutin and acetylated arbutin were found to exert pro-apoptotic effects on B16 murine melanoma cells, mediated through the mitochondrial pathway. The findings of the present study also support the use of acetylated arbutin as a new potential candidate agent for skin whitening and melanoma treatment.