Ceramide is a bioactive sphingolipid that mediates a variety of cell functions. However, the effects of ceramide on cell growth and the melanogenesis of melanocytes are not known. In the present study, we investigated the actions of cell-permeable ceramide and its possible role in the signaling pathway of a spontaneously immortalized mouse melanocyte cell line, Mel-Ab. Our results show that C2-ceramide inhibits DNA synthesis in Mel-Ab cells and G361 human melanoma cells in a dose-dependent manner. Cell cycle analysis confirmed the inhibition of DNA synthesis by a reduction in the S phase. To investigate the ceramide signaling pathway, we studied whether C2-ceramide is able to influence extracellular signal-regulated kinase (ERK) and/or Akt/protein kinase B (PKB) activation. We demonstrated that phosphorylated Akt/PKB is decreased by C2-ceramide, whereas phosphorylated ERK was only slightly affected. Therefore, the C2-ceramide-induced inactivation of Akt/PKB may be closely related to the reduced cell proliferation of Mel-Ab cells. Furthermore, we assessed the effects of C2-ceramide on the pigmentation of Mel-Ab cells. The results obtained showed that the melanin content of cells was significantly reduced by C2-ceramide at concentrations in the range of 1-10 μM, and that the pigmentation-inhibiting effect of C2-ceramide is much greater than that of kojic acid at 1-100 μM. In addition, we found that the activity of tyrosinase is reduced by C2-ceramide treatment. Our results demonstrate that C2-ceramide reduces the pigmentation of Mel-Ab cells by inhibiting tyrosinase activity.