Mammary tumor cells are required to degrade the surrounding matrix and disseminate in order to metastasize, and both of these processes are controlled by a tumor cell-signaling network that remains poorly defined. MEKK1 is a MAPKKK that regulates both the extracellular signal regulated kinase (ERK1/2) and the c-Jun amino terminal kinase (JNK) signaling pathways. MEKK1 signaling regulates migration through control of cell adhesion and is required for inducible expression of urokinase-type plasminogen activator (uPA). MEKK1-deficient mice with mammary gland-targeted expression of the polyoma middle T antigen (PyMT) transgene develop primary mammary tumors at a rate and frequency similar to wild-type littermates, indicating that MEKK1 deficiency does not affect PyMT-mediated transformation. However, MEKK1−/− mice display significantly delayed tumor cell dissemination and lung metastasis. Delayed MEKK1-dependent tumor dissemination is associated with markedly reduced tumor uPA expression, gelatinase activity, and prolonged tumor basement membrane integrity. siRNA-mediated MEKK1 knockdown inhibits uPA activity, cell migration and invasion in MDA-MB-231 human breast cancer cells. Thus MEKK1 controls tumor progression by regulating both the migration and proteolysis aspects of tumor cell invasiveness. To our knowledge, this is the first example of a MAPKKK that regulates metastasis through control of tumor invasiveness.