Glycogen synthase kinase 3β (GSK3β) is implicated in many biological events, including embryonic development, cell differentiation, apoptosis, and the insulin response. GSK3β also plays a key role in the Wnt/β-catenin pathway. The master regulator of the pigmentation microphthalmia-associated transcription factor (MITF) is a target for the Wnt pathway, however, to date, the regulatory role of GSK3β in the control of melanogenesis has not been elucidated. In this study, we evaluated the effect of inhibiting GSK3β activity on the regulation of melanocyte differentiation. Exposure of the murine melanoma cell line B16 and normal human melanocytes to GSK3β specific inhibitors (SB216763, SB415286, BIO, and LiCl) resulted in a dose-dependent accumulation of β-catenin. This is associated with the induction of melanocyte differentiation-associated markers such as melanin synthesis, tyrosinase activity, and expression of tyrosinase and the microphthalmia-associated transcription factor. Attenuation of GSK3β activity has an inhibitory effect on cell growth, and this was accompanied by morphological changes. Moreover, treatment of B16 cells with a siRNA targeted against β-catenin completely abolished the promelanogenic effect of GSK3β inhibition, however, the overexpression of a constitutively active mutant form of β-catenin (pCS2β-cat-mut) only slightly increased the degree of pigmentation. These results demonstrated that GSK3β is implicated in the regulation of melanogenesis and that pharmacological inhibition of its activity could increase melanin synthesis through mechanisms probably not restricted to Wnt/β-catenin pathway activation.