The microphthalmia-associated transcription factor (MITF) is a pivotal regulator of melanogenic enzymes for melanogenesis, and its expression is modulated by many transcriptional factors at the transcriptional level or post-transcriptional level through microRNAs (miRNAs). Although several miRNAs modulate melanogenic activities, there is no evidence of their direct action on MITF expression. Out of eight miRNAs targeting the 3'-UTR of Mitf predicted by bioinformatic programs, our results show miR-218 to be a novel candidate for direct action on MITF expression. Ectopic miR-218 dramatically reduced MITF expression, suppressed tyrosinase activity, and induced depigmentation in murine immortalized melan-a melanocytes. MiR-218 also suppressed melanogenesis in human pigmented skin organotypic culture (OTC) through the repression of MITF. An inverse correlation between MITF and miR-218 expression was found in human primary skin melanocytes and melanoma cell lines. Taken together, our findings demonstrate a novel mechanism involving miR-218 in the regulation of the MITF pigmentary process and its potential application for skin whitening therapy.