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Vitiligo is a common depigmentary disease characterized as diagnosis simplicity and cure difficulty in view of the ambiguity of etiology, thus novel and effective treatments are urgently needed. Paeoniflorin, the major active compound extracted from the root of Paeonia lactiflora Pall, a traditional Chinese medicine, has been validated pharmacological properties such as antioxidant stress, a theory participating in the occurrence of vitiligo, but the effect on melanogenesis is still unclear. In this study, melanosythesis effect of paeoniflorin and the potential mechanism were evaluated. We found that treatment with paeoniflorin at the concentration of 10 μg/ml significantly increased melanin content and intracellular tyrosinase activity of human melanocytes, in accordance with the elevation of protein levels of microphthalmia-associated transcription factor (MITF), tyrosinase-related protein 1 (TRP-1). In addition, we also investigated that paeoniflorin promoted phosphorylation of cAMP-response element binding (CREB) and extracellular signal-regulated kinase (ERK) without affecting p38 and c-Jun N-terminal kinase (JNK). These results demonstrated that paeoniflorin had a synergistic effect on normal human melanocytes via ERK/CREB pathway with up-regulation of MITF and TRP-1, enhancing melanin synthesis. Meanwhile, the milder pathological changes in vitiligo mice treat with paeoniflorin also confirmed its potential in treating vitiligo. To sum up, we suggest that paeoniflorin may be a potential medicine of vitiligo treatment in clinical.