β-Lapachone is an ortho naphthoquinone obtained from the bark of the lapacho tree (Tabebuia avellanedae), which has been used medicinally for centuries. The purpose of this study was to investigate the effects of β-lapachone on inhibitory mechanism of melanogenesis. β-Lapachone inhibited melanin synthesis and tyrosinase activity at 0.8 μM in melan-a cells. Also, β-lapachone reduced the expression of tyrosinase and tyrosinase-related protein-1 at transcriptional and translational levels. The decreased expression of tyrosinase and tyrosinase-related protein-1 might result from the reduced microphthalmia-associated transcription factor (MITF) level which regulates major melanogenic proteins. The reduced level of MITF was associated with delayed ERK activation by β-lapachone. Furthermore, β-lapachone reduced melanogenesis in the human 3D skin tissue culture; besides, it dramatically inhibited body pigmentation of zebrafish and decreased melanin content and tyrosinase activity. These results show that β-lapachone may be useful as a potential depigmentation agent for various hyperpigmentation disorders.