Melanin plays a crucial role in camouflage, social communication and protection against harmful ultraviolet radiations. Melanin is a synthesized by melanocytes through melanogenesis and several intrinsic and extrinsic factors are involved during the process. Any change occur in normal melanogenesis process can cause severe pigmentation problems of hypopigmentation or hyperpigmentation. The present study is based on evaluation of the effect of thymoquinone on melanogenesis and their possible mechanism of action using B16F10 melanoma cell line for the production via blocking signaling pathways. Phase contrast microscopy, cell viability, tyrosinase activity, melanin content and western blot analysis were use in the present study. In the present investigation cultured melanocytes exhibit that the stimulation of melanin synthesis when treated with thymoquinone. Tyrosinase activity and melanin production in B16F10 melanoma cell line were increased in doze-dependent manner. In western blot we investigated the involvement of the cAMP/PKA pathway in thymoquinone induced melanogenesis. It was observed protein kinase inhibitors PKA, PKC, PKB and MEK1 decreased the stimulatory effects of thymoquinone from 11.45- fold value to 8.312, 6.631, 4.51, and 7.211-fold value, respectively. However, the results also prove that thymoquinone may partially induced tyrosinase expression via PKA, PKB, PKC and MEK1 signaling pathways. Present finding proposed that thymoquinone is a protective challenger for melanogenesis and it might be useful for the treatment of hypopigmentary disorders.