Homeoproteins are transcription factors that act as master regulators of development and are frequently dysregulated in cancers. During embryogenesis, the Six1 homeoprotein is essential for the expansion of precursor cell populations that give rise to muscle and kidney, among other organs. Six1 overexpression is observed in numerous cancers, resulting in increased proliferation, survival, and metastasis. Here, we investigate whether Six1 can play a causal role in mammary tumor initiation. We show that Six1 overexpression in MCF12A mammary epithelial cells promotes multiple properties associated with malignant transformation, including increased proliferation, genomic instability, and anchorage-independent growth. We further show that this transformation is dependent on up-regulation of its transcriptional target, cyclin A1, which is normally expressed in the embryonic mammary gland but dramatically reduced in the adult gland. Six1-transformed MCF12A cells are tumorigenic in nude mice, forming aggressive tumors that are locally invasive and exhibit peritumoral lymphovascular invasion. In human breast carcinomas, expression of Six1 and cyclin A1 mRNA correlate strongly with each other (P < 0.0001), and expression of Six1 and cyclin A1 each correlate with Ki67, a marker of proliferation (P < 0.0001 and P = 0.014, respectively). Together, our data indicate that Six1 overexpression is sufficient for malignant transformation of immortalized, nontumorigenic mammary epithelial cells, and suggest that the mechanism of this transformation involves inappropriate reexpression of cyclin A1 in the adult mammary gland. [Cancer Res 2008;68(7):2204–13]