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Skin color is determined by the amount of melanin present. Many enzymes have a relationship in melanin synthesis. Especially, tyrosinase is well known as key enzyme for melanin synthesis. MITF (Microphthalmia-associated transcription factor) belongs to the basic helix-loop-helix-zip family of transcription factors and is the major regulator of tyrosinase and other related enzymes (TRPs) by binding E-box (CATGTG). To screen for MITF – E-box binding inhibitor, EMSA (Electrophoretic Mobility Shift Assay) was performed. Based on the computer-simulated structure, 27 chemicals were selected and were investigated as MITF – E-box binding inhibitors. To check the depigmenting activity, cell tests were performed by using melan-a cells. Among them, compound #18 was found to show the most potent inhibitory activity against MITF – E-box binding. As a result of EMSA, intensities of MITF – E-box bands were reduced by compound #18 in a dose-dependent manner. In addition, unlabelled Tyr-p, mutated Tyr-p and random oligomer were used for EMSA to investigate the competitive binding inhibitory effect. As a result, excess unlabelled Tyr-p was an effective binding competitor. Conversely, mutated Tyr-p and random oligomer did not compete with MITF. To check the protein level of MITF, tyrosinase, and TRP-1, western blot analysis was performed by using melan-a cells. The protein level of tyrosinase was reduced by compound #18, but MITF and TRP-1 were not reduced. Therefore, compound #18 could be used as a specific MITF – E-box binding inhibitor on melanocytes.