Two natural acetophenone derivatives, 2,5-dihydroxyacetophenone (2,5-DHAP) and 2,6-DHAP, were purified from Cynanchum bungei and identified as murine tyrosinase inhibitors. Investigation into 2,5-DHAP showed it to be an uncompetitive inhibitor of murine tyrosinase (KI 0.28 mm). 2,5-DHAP strongly inhibited both melanogenesis and cellular tyrosinase activity in vitro in 3-isobutyl-1-methylxanthin-stimulated B16 mouse melanoma cells or in vivo in zebrafish and mouse models, but showed no cytotoxicity at the concentrations used. In B16 cells, 2,5-DHAP inhibition was dose-dependent and was fourfold greater than that of arbutin. 2,5-DHAP had no effect on the expression of tyrosinase protein or mRNA, as confirmed by Western blotting and quantitative real-time reverse transcription polymerase chain reaction, respectively. A 2% gel preparation of 2,5-DHAP applied to the skin of mice significantly increased the average skin-whitening index (L value), indicating its potential use as a treatment for skin hyperpigmentation in humans.