The complex and diverse role of Rab5 isoforms in endocytic transport is highlighted by the large number of proteins with which they interact. Rab5 is activated by as many as six GEFs including Rabex5, Gapex-5, Rin1, Rin2, Rin3 and Als2 and deactivated by at least two Rab5 GTPaseactivating proteins RabGap-5 and RN-Tre. Rab5 effectors include EEA1 and Rabinosyn-5, proteins that mediate Rab tethering to membranes through the well-characterized FYVE domain, and APPL1 and APPL2, proteins that interact with Rab5 to orchestrate membrane trafficking and that affect gene transcription. PI3 kinase and PI5 and PI4 phosphatases interact with Rab5 to regulate aspects of signal transduction and the temporal regulation of phosphoinositide turnover required for progression of cargo through the early endocytic pathway. This collection of Rab5 isoform effectors, GEFs and GAPs form a large interactive network that orchestrates and regulates the multiple functions associated with the early endosomal compartment. The current study builds on our earlier work on Rab5 isoform specificity and on the work of Palamidessi el.al, who showed that Rab5 and the Rab5 GAP, RN-Tre, modulates Rac activity and cell motility. Here, we examined the differential effects of Rab5 isoform silencing on cell motility. We report that Rab5C, but neither Rab5A nor Rab5B, is selectively associated with the growth factor-activation of Rac1 and with enhanced cell motility. The current study builds on earlier work showing that Rab5A selectively regulates growth factor receptor trafficking and focuses on the role of Rab5C in selectively regulating cell motility and cytoskeletal dynamics. DiFiore and colleagues have suggested that Rab5 acts as a critical switch in the endocytic circuitry by which Rac1 can be activated and re-routed to specific sites at the plasma membrane to initiate actin assembly. More recently, the same group has demonstrated that the Rab5 GAP RN-Tre, delays the turnover of focal adhesions clearly indicating a role for Rab5 in cell migration. In their study, Rab5 was examined by silencing all three Rab5 isoforms. Here we show that Rab5C preferentially serves this function via kinase inhibitors modulating a combination of signaling and trafficking pathways. The correlation of Rac1 activation with Rab5 isoform silencing/over-expression was tested both at steady state and under EGF stimulation. We found that all three Rab5 isoforms were capable of potentiating Rac1 activity following exogenous expression. On the contrary, Rac1 activation responded to the individual depletion of endogenous Rab5 isoforms very differently. Loss of Rab5C function suppressed Rac1 activity both at steady state and when stimulated by EGF. Rab5B depletion showed only mild suppression. In correspondence with the reduced Rac1 activity, Rab5Cdepleted cells exhibited altered cell shape and defective locomotion towards open wound space in a scratch-wound assay or in a transwell migration assay with a serum gradient. These findings suggest that Rab5C plays a preferential role in Rac1-mediated cell migration. In contrast to over-expression, depletion of Rab5A mildly increased Rac1 activity and cell motility.