Poly-gamma-glutamate (gamma-PGA) has been considered as one of the most promising biomaterials with a wide range of applications, but there has been no report that directly shows the anti-tyrosinase and anti-melanogenesis properties of gamma-PGA. In the present study, we investigated the inhibitory effects of gamma-PGA with low molecular weight (Mw; l gamma-PGA) and high Mw (h gamma-PGA) on mushroom tyrosinase and murine tyrosinase activities and on melanogenesis in B16 melanoma cells. First, we showed that both l gamma-PGA and h gamma-PGA could effectively inhibit mushroom tyrosinase activities including monophenolase and diphenolase activities in a dose-dependent manner. Second, both l gamma-PGA and h gamma-PGA showed strong anti-tyrosinase activity and anti-melanogenesis in B16 melanoma cells. Third, both l gamma-PGA and h gamma-PGA inhibited forskolin-induced tyrosinase activity and melanogenesis by decreasing the levels of intracellular reactive oxygen species and nitric oxide while increasing the catalase activity in B16 cells. This is the first report on the anti-melanogenesis effect of gamma-PGA, which suggests that gamma-PGA could have a potential in the cosmetic skin whitening business, therapeutic applications and the food industry.