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Background and Purpose cAMP as a second messenger stimulates expression of microphthalmia-associated transcription factor (MITF) or the tyrosinase gene in UVB-induced skin pigmentation. Diphenylmethylene hydrazinecarbothioamide (QNT 3-80) inhibits α-melanocyte-stimulating hormone (α-MSH)-induced melanin production in B16 murine melanoma cells but its molecular basis remains to be defined. Here, we investigated the mechanism underlying the amelioration of skin hyperpigmentation by QNT 3-80. Experimental Approach We used melanocyte cultures with raised levels of cAMP and UVB-irradiated dorsal skin of guinea pigs for pigmentation assays. Immunoprecipitation, kemptide phosphorylation, fluorescence analysis and docking simulation were applied to elucidate a molecular mechanism of QNT 3-80. Key Results QNT 3-80 inhibited melanin production in melanocyte cultures with elevated levels of cAMP, including those from human foreskin. This compound also ameliorated hyperpigmentation in vivo in UVB-irradiated dorsal skin of guinea pigs. As a mechanism, QNT 3-80 directly antagonized cAMP binding to the regulatory subunit of PKA, nullified the dissociation and activation of inactive PKA holoenzyme in melanocytes and fitted into the cAMP-binding site on the crystal structure of human PKA under the most energetically favourable simulation. QNT 3-80 consequently inhibited cAMP- or UVB-induced phosphorylation (activation) of cAMP-responsive element-binding protein in vitro and in vivo, thus down-regulating expression of genes for MITF or tyrosinase in the melanogenic process. Conclusions and Implications Our data suggested that QNT 3-80 could contribute significantly to the treatment of skin disorders with hyperpigmented patches with the cAMP-binding site of PKA as its molecular target.