Abstract: Objective To study the relationship between Nanog-promoted metastasis of breast cancer and ezrinT567 phosphorylation, and explore the possible mechanism by which Nanog regulates ezrinT567 phosphorylation. Methods A siRNA construct targeting Nanog was transfected in breast cancer cells to knock down Nanog expression, and the changes in the cell invasion was detected using Transwell assay. The expression levels of Nanog and PKC and the phosphorylation level of ezrinT567 were detected using Western blotting and immunofluorescent staining; the protein interaction between PKCɛ and ezrin was assayed by co-immunoprecipitation and Western blotting. Results Nanog knockdown significantly decreased the expression of PKCɛ protein, phosphorylation level of ezrinT567 and the invasion ability of breast cancer cells. PKCε knockdown obviously decreased the phosphorylation level of ezrinT567 in the cells, and PKCɛ and ezrin were co-immunoprecipitated. Concludions Nanogcan can upregulate the expression of PKCɛ to promote the phosphorylation of ezrinT567, which can be a new mechanism by which Nanog promotes tumor metastasis.