The Six1 homeoprotein is an important mediator of normal development, where it is critical for the proliferation of precursor cell populations that ultimately constitute the muscle, kidney and inner ear, among other organs. Interestingly, its overexpression has been observed in numerous cancers, where it contributes to the proliferative and metastatic ability of the cancer cells. Here we show that Six1 not only regulates the cell cycle, but is itself regulated throughout the cell cycle via ubiquitin-mediated proteolysis. The protein is present from the G(1)/S boundary until mitosis, when it is degraded via the anaphase-promoting complex (APC) with its activating subunit Cdh1. However, unlike most identified APC(Cdh1) targets, Six1 does not contain functional destruction or KEN box motifs that are necessary for its degradation. Instead, the Six1 protein contains multiple, as yet undefined, sequences within its N- and C-termini responsible for its degradation, including an N-terminal region that binds to Cdh1. Cell cycle regulation of Six1 occurs both transcriptionally and post-translationally via phosphorylation; therefore, this study demonstrates a third and novel mechanism of cell cycle-specific regulation of Six1, underscoring the importance of confining its activity to a defined cell cycle window from the G(1)/S boundary to early mitosis.