The 18-kDa translocator protein (TSPO) is highly conserved among different species but with perplexing biochemical function. Multiple ligands of TSPO show commendable regulatory activities among lots of biological functions, such as neuroinflammation, cholesterol transport and so on. These researches support that TSPO may be a potential target for disease treatment and drug development. Previous studies have shown that its ligand agonist benzodiazepines have the effect of promoting melanin, but the specific mechanism is still unclear. In this research, we firstly confirmed the melanogenesis promotion of Ro5-4864 in mouse melanoma cell line, human skin tissue and zebrafish embryos through inducing melanin synthesis and melanosome transport. Molecular genetics and pharmacological studies showed that TSPO deficiency did not affect melanin production in B16F10 cells and zebrafish embryos, nor did it affect melanin promotion effect of Ro5-4864. The expression of lots of melanogenesis related proteins, such as TYR, TRP-1, DCT, Mlph and Rab27 were upregulated in Ro5-4864 treatment cell with or without Tspo. These results indicated that Ro5-4864 induced melanogenesis in a TSPO independent manner. However, further research is still needed to clarify the direct downstream target of Ro5-4864 in promoting melanogenesis.