Six1 is a critical regulator of embryonic development that requires interaction with the Eya family of proteins (Eya1-4) to activate the transcription of genes involved in neurogenesis, myogenesis and nephrogenesis. Although expression of Six1 and Eya family members is predominantly observed in development, their overexpression is observed in numerous cancers. Importantly, both Six1 and Eya have independently been shown to mediate breast cancer metastasis, but whether they functionally interact during tumor progression has not been explored. Herein, we demonstrate that knockdown of Eya2 in MCF7 mammary carcinoma cells reverses the ability of Six1 to induce transforming growth factor-β signaling, as well as to induce characteristics associated with epithelial-mesenchymal transition and cancer stem cells, suggesting that Six1 is dependent on Eya2 to mediate numerous pro-metastatic characteristics. The importance of the Six1-Eya interaction in human breast cancer is underscored by the finding that high levels of Six1 correlate with shortened time to relapse and metastasis as well as decreased survival only when co-expressed with high levels of Eya2. Overall, these data implicate Eya2 as a necessary co-factor for many of the metastasis promoting functions of Six1, suggesting that targeting the Six1-Eya interaction may inhibit breast cancer progression. As Six1 and Eya2 are not highly expressed in most adult tissues, the Six1-Eya interaction may be a valuable future therapeutic target whose inhibition would be expected to impair breast cancer progression while conferring limited side effects.