AbstractRac1 and Cdc42 are members of the Rho family of small GTPases that play essential roles in diverse cellular functions, including cell migration. The activities of these Rho family proteins are controlled by growth factor receptor activation and cell‐ECM interactions. Here, we show that maspin, a well‐documented tumor suppressor gene, also controls cell motility through inhibiting Rac1/Cdc42 activity. Using the GST‐PAK and GST‐Rho binding protein pull‐down assays for GTP‐bound Rac1, Cdc42, and RhoA, we showed that treatment of MDA‐MB‐231 tumor cells with recombinant maspin for a short time period significantly inhibited the activity of Rac1 and Cdc42, but not RhoA. The reactive site loop (RSL) within maspin protein is the functional domain involved in the inhibition. Maspin mutants with the RSL deleted or a point mutation in the RSL region lost their inhibitory activity. We further examined the ability of maspin to inhibit Rac1‐ and Cdc42‐mediated signaling pathways and transcription factors. Treatment of MDA‐MB‐231 cells with maspin led to the inhibition of JNK kinase activity as assayed by immuno‐kinase assays. In addition, the AP‐1 transcription activity downstream of JNK kinase pathway was also reduced. Together, we have identified Rac1 and Cdc42 as the downstream targets that mediate the inhibition of mammary tumor cell migration by maspin. Cell Motil. Cytoskeleton 2007. © 2007 Wiley‐Liss, Inc.