The ERM proteins ezrin, radixin and moesin are adaptor proteins that link plasma membrane receptors to the actin cytoskeleton. Ezrin and moesin have been implicated in cell polarization and cell migration, but little is known about the involvement of radixin in these processes. Here we show that radixin is required for migration of PC3 prostate cancer cells, and that radixin, but not ezrin or moesin, depletion by RNA interference increases cell spread area and cell-cell adhesion mediated by adherens junctions. Radixin depletion also alters actin organization, and distribution of active phosphorylated ezrin and moesin. Similar effects were observed in MDA-MB-231 breast cancer cells. The phenotype of radixin-depleted cells is similar to that induced by constitutively active Rac1, and Rac1 is required for the radixin knockdown phenotype. Radixin depletion also increases the activity of Rac1 but not Cdc42 or RhoA. Analysis of Rac guanine nucleotide exchange factors (GEFs) suggests that radixin affects the activity of Vav GEFs. Indeed, Vav GEF depletion reverses the phenotype of radixin knockdown and reduces the effect of radixin knockdown on Rac1 activity. Our results indicate that radixin plays an important role in promoting cell migration by regulating Rac1-mediated epithelial polarity and formation of adherens junctions through Vav GEFs.