Abstract The forkhead box transcription factor FOXD3 is a stemness factor that prevents the production of melanocyte progenitors from the developing neural crest; however, its role in human cancers is not known. Transformation of melanocytes gives rise to melanoma. In two thirds of melanomas, the serine/threonine kinase B-RAF is mutated to a constitutively active form. Here, we show that FOXD3 levels are upregulated following attenuation of B-RAF and mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK) signaling in mutant B-RAF harboring human melanoma cells. This effect was selective because FOXD3 was not upregulated following MEK inhibition in wild-type B-RAF melanoma cells and mutant B-RAF thyroid carcinoma cells. Ectopic FOXD3 expression potently inhibited melanoma cell growth without altering mutant B-RAF activation of ERK1/2. Inhibition of cell growth was due to a potent G1 cell cycle arrest and was associated with p53-dependent upregulation of p21Cip1. FOXD3-induced cell cycle arrest was prevented by p53 depletion and, to a lesser extent, p21Cip1 depletion. These studies show that FOXD3 is suppressed by B-RAF, uncover a novel role and mechanism for FOXD3 as a negative cell cycle regulator, and have implications for the repression of melanocytic lineage cells. Cancer Res; 70(7); 2891–900