Melanin, the principal component of skin pigmentation, is produced through tyrosinase activity. Recently, supersulphides have been identified in human cells and found to play a role in maintaining cellular activities. Glutathione trisulphide (GSSSG), a supersulphide with antioxidant properties, has various biological effects. However, the influence of melanin production on intracellular persulphide levels remains unclear, and no studies have reported the anti-melanogenic effects of GSSSG. Therefore, we aimed to elucidate the impact of melanogenesis on intracellular persulphide abundance and synthesis, as well as the efficacy of GSSSG in inhibiting melanogenesis in melanoma cells. In this study, B16-F0 cells were used to evaluate the melanin and intracellular persulphide levels by alpha-melanocyte-stimulating hormone (α-MSH). Moreover, the effects of GSSSG on melanin production were studied. The results revealed that α-MSH-induced melanogenesis significantly increased melanin production and decreased intracellular persulphide levels. Furthermore, the expression of persulphide synthesis genes, including Cars2 and Cbs, was significantly downregulated by α-MSH. In contrast, GSSSG significantly suppressed α-MSH-induced melanin production. Notably, GSSSG restored the intracellular persulphide levels reduced by α-MSH and upregulated Cars2 and Cbs expression. These findings suggest that GSSSG exerts anti-melanogenesis effects, maintains intracellular persulphide levels, and improves skin brightness.