The relationship of carcinogenesis and DNA methyltransferases has attracted extensive attention in tumor research.We reported previously that inhibition of de novo DNA methyltransferase 3a (Dnmt3a) in murine B16 melanoma cells significantly suppressed tumor growth and metastasis in xenografted mouse model.Here,we further demonstrated that knockdown of Dnmt3a enhanced the proliferation in anchor-independent conditions of B16 cells,but severely disrupted its multipotent differentiation capacity in vitro.Furthermore,transforming growth factor β1,a key trigger in stem cell differentiation and tumor cell epithelial-mesenchymal transition (EMT),mainly induced apoptosis,but not EMT in Dnmt3a-deficient B16 cells.These data suggested that Dnmt3a is required for maintaining the tumor stemness of B16 cells and it assists B16 cells to escape from death during cell differentiation.Thus it is hypothesized that not only extraordinary selfrenewal ability,but also the capacity of multipotent differentiation is necessary for the melanoma tumorigenesis.Inhibition of multipotent differentiation of tumor cells may shed light on the tumor treatment.