Vitiligo is a depigmentation disorder of the skin. It is primarily caused by the destruction of melanocytes or obstruction of the melanin synthesis pathway. Melanin is a type of skin pigment that determines skin color. The seeds of Vernonia anthelmintica (L.) Willd (Kaliziri) are used for treating skin diseases including vitiligo in traditional Uyghur medicine. 1,5-Dicaffeoylquinic acid (1,5-diCQA) is a natural polyphenolic compound widely distributed in plants and extracted from Kaliziri seeds. Therefore, in the present study, the effect of 1,5-diCQA on melanin synthesis in B16 cell was evaluated, and its molecular mechanism was explored. The results indicated that 1,5-diCQA treatment of B16 cells stimulated an increase of intracellular melanin level and tyrosinase (TYR) activity without cytotoxicity. Reverse transcription quantitative polymerase chain reaction results also indicated that 1,5-diCQA may markedly improve the protein expression and RNA transcription of microphthalmia-associated transcription factor (MITF), melanogenic enzyme Tyr, tyrosinase-related protein 1 (TRP 1) and tyrosinase-related protein 2 (TRP 2). Additional results identified that 1,5-diCQA may promote the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signal-regulated kinase (ERK) MAPK. Notably, the increased levels of intracellular melanin synthesis and tyrosinase expression induced by 1,5-diCQA treatment were significantly attenuated by the protein kinase A (PKA) inhibitor H-89. Intracellular cyclic adenosine monophosphate (cAMP) concentration and phosphorylation of cAMP-response element binding protein was increased following 1,5-diCQA treatment. These results indicated that 1,5-diCQA stimulated melanogenesis via the MAPK and cAMP/PKA signaling pathways in B16 cells, which has potential therapeutic implications for vitiligo.