The phosphatase and transactivator EYA family proteins are overexpressed in many cancer cell lines and are abundantly distributed in undifferentiated cells during development. Loss-of-function studies have shown that EYA1 is required for cell proliferation and survival during mammalian organogenesis. However, how EYA1 is regulated during development is unknown. Here, we report that EYA1 is regulated throughout the cell cycle via ubiquitin-mediated proteolysis. The level of EYA1 protein fluctuates in the cell cycle, peaking during mitosis and dropping drastically as cells exit into G(1). We found that EYA1 is efficiently degraded during mitotic exit in a Cdh1-dependent manner and that these two proteins physically interact. Overexpression of Cdh1 reduces the protein levels of ectopically expressed or endogenous EYA1, whereas depletion of Cdh1 by RNA interference stabilizes the EYA1 protein. Together, our results indicate that anaphase-promoting complex/cyclosome (APC/C)-Cdh1 specifically targets EYA1 for degradation during M-to-G(1) transition, failure of which may compromise cell proliferation and survival.