Insulin-like growth factor binding protein 1 (IGFBP1), the main secretory product of the decidualized endometrium of a pregnant woman, has previously been shown to interact with the alpha₅beta₁ integrin of extravillous trophoblast (EVT) cell surface to stimulate its migration in an IGF-independent manner. This migration stimulation has also been shown to require activation of extracellular signal regulated kinases 1 and 2 (ERK1/2; mitogen-activated protein kinase [MAPK] 3/1]) and focal adhesion kinase. The present study examined the roles of Rho GTPases RHOA, RHOC, RAC1, and CDC42 as well as RHO kinases ROCK1 and ROCK2 in IGFBP1-mediated migration of an immortalized EVT cell line HTR-8/SVneo. A nonselective RHO kinase inhibitor, Y27632, as well as siRNAs selective for ROCK1 and ROCK2 decreased the migration of these cells in a Transwell migration assay, and this inhibition could not be restored by IGFBP1. Clostridium difficile toxin B, which inhibits all the Rho GTPases, RAC inhibitor NSC23766, RAC1 siRNA, and CDC42 siRNA, decreased their basal migration, but none of these inhibitions except CDC42 siRNA-induced inhibition could be restored by IGFBP1. Clostridium botulinum C3 exoenzyme that inhibits RHOA, RHOB, and RHOC inhibited basal migration but not IGFBP1-induced migration. IGFBP1-induced activation of ERK1/2 (MAPK3/1), which did not require RHO proteins, might function as an alternate pathway for RHO action. However, selective siRNA-mediated downregulation of RHOA inhibited basal, but not IGFBP1-mediated, migration, whereas that of RHOC inhibited both basal and IGFBP1-mediated migration of these EVT cells. Therefore, RHO kinase, RHOC, and RAC1 are essential, but RHOA and CDC42 are not essential, for IGFBP1-induced EVT migration.