Although Wnt1 downstream signaling components have been well studied and activated in human cancer, the pathways that regulate Wnt1 itself have not been explored in depth. Here, we provide gain-of-function, loss-of function, and molecular evidence supporting functional interactions between metastasis-associated protein 1 short-form (MTA1s), metastasis-associated protein 1 (MTA1), and Wnt1 signaling components during mammary gland development and tumorigenesis. Using multiple model systems involving overexpression or knockdown of MTA1s or MTA1, we discovered that MTA1s and MTA1 hyperactivate the Wnt1 pathway due to increased expression of Wnt1 transcription. MTA1s and MTA1 physically interact with Six3 chromatin, a protein product of which is a direct histone deacetylase inhibitor-dependent repressor of Wnt1 transcription. Deletion of the MTA1s and MTA1 allele in murine embryonic fibroblasts resulted in the upregulation of Six3 and downregulation of Wnt signaling. In addition, mammary glands from the MTA1s/MTA1-/- mice exhibited increased recruitment of Six3 corepressor complex to the Wnt1 promoter and inhibition of Wnt1 pathway in mammary glands. These findings identify MTA1s and MTA1 as important upstream modifiers of the Wnt1 transcription, and consequently its functions, by directly inhibiting the transcription of Six3, allowing derepression of Wnt1 transcription. ©2010 AACR.