CDC25 phosphatases are not only rate-limiting activators of cyclin-dependent kinases (CDKs) but also important targets of the CHK1/CHK2-mediated checkpoint pathway. Each isoform of the mammalian CDC25 family seems to exert unique biological functions. CDC25A is a critical regulator for both G1-S and G2-M transitions and essential for embryonic cell proliferation after the blastocyst stage. CDC25B is dispensable for embryogenesis but required for meiotic progression of oocytes in a manner analogous to Drosophila Twine or C. elegans cdc-25.1. Moreover, CDC25A and CDC25B appear to regulate different events or stages of mitosis. CDC25B may mediate the activation of CDK1/Cyclin B at the centrosome during prophase, while CDC25A may be required for the subsequent full activation of nuclear CDK1/Cyclin B. CDC25C is dispensable for both mitotic and meiotic divisions, although it is highly regulated during the processes. Excessive levels of CDC25A and CDC25B are often observed in various human cancer tissues. Deregulated expression of these phosphatases allows cells to overcome DNA damage-induced checkpoint, leading to genomic instability. Studies using mouse models demonstrated that deregulated expression of CDC25A significantly promotes RAS- or NEU-induced mammary tumor development with chromosomal aberrations, whereas decreased CDC25A expression in heterozygous knockout mice delays tumorigenesis. These biological properties of CDC25 phosphatases provide significant insight into the pathobiology of cancer and scientific foundation for anti-CDC25 therapeutic intervention.