2,3,5,6-Tetramethylpyrazine (TMP) is known as a composition of Ephedra sinica and it has been used in the treatment of several disorders such as asthma, heart failure, rhinitis, and urinary incontinence. It has been reported that TMP inhibits melanoma metastasis and suppression angiogenesis by VEGF. The inhibitory activity of melanogenic proteins by TMP was confirmed in UVA-induced melanoma/keratinocyte co-culture system in this paper. The melanin content, cell viability and cytokines release such as TNFα, IL-1β, IL-8 and GM-CSF were measured by ELISA assay. In addition, TRP1, MITF and MAPK signaling protein expression were also evaluated by Western blotting analysis. Decreasing melanogenic factors (TRP1, MITF, and MAPK) and factors (TNFα, IL-1β, IL-8, and GM-CSF) improving skin cancer and inflammation were identified. It suggests that TMP can serve as a potent candidate for regulation of melanogenesis.