Urokinase-type plasminogen activator (uPA) and its receptor, uPAR, play important roles in promoting cancer cell adhesion, migration and invasion. Rho GTPases are key coordinators of these processes; the Rho GTPase Rac1 has previously been implicated in uPA- and/or uPAR-induced migratory or morphological cell responses. We used RNAi to deplete 12 different Rho GTPases to screen for effects on uPA-stimulated migration, and found that depletion of RhoB significantly reduces uPA-induced migration and invasion of prostate carcinoma cells. RhoB depletion did not affect the expression or surface levels of uPAR but reduced the uPAR-induced increase in levels of several integrins and inhibited uPAR signalling to the actin regulator cofilin, the cell-adhesion signal-transduction adaptor molecule paxillin and the serine/threonine kinase Akt. uPAR rapidly activated RhoB and increased RhoB expression. RhoB depletion also reduced cell adhesion to and spreading on vitronectin, which is a uPAR ligand. This correlated with decreased association between integrins and uPAR and reduced integrin β1 activity. Our results indicate that RhoB is a key regulator of uPAR signalling in cell adhesion, migration and invasion.