The control of melanogenesis is an important strategy in the treatment of abnormal skin pigmentation for cosmetic purpose. In mammals, melanin synthesis is regulated by melanogenic enzymes, such as tyrosinase, tyrosinase related protein (TRP)-1 and TRP -2. Our aim was to study whether Pini Nodi Lignum (PNL), an extract of Pinus tabulaeformis nodes, possesses antimelanogenic activity. To examine the inhibitory effect of PNL on melanogenesis, tyrosinase inhibition assay, melanin content assay, RT-PCR analysis and Western blotting of tyrosinase, TRP-1, and TRP-2 were performed using HM3KO melanoma cells. The results showed that treatment of HM3KO cells with PNL inhibited melanin formation without affecting normal cell growth. Cell-free tyrosinase inhibition assay exhibited that PNL directly inhibited mushroom tyrosinase enzyme catalytic activity. Moreover, it was found that application of PNL significantly suppressed tyrosinase and TRP-1 mRNA and protein expression, but not that of TRP-2, in HM3KO cells. These results suggest that the inhibitory effect of PNL on melanogenesis is not only by inhibiting tyrosinase catalytic activity but also by inhibiting expression of melanogenic proteins at the transcriptional and translational levels. The PNL can be exploited as the effective whitening agent that possesses antimelanogenic activity.