Rho GTPases and the dynamic assembly and disassembly of actin filaments have been shown to have critical roles in both the internalization and trafficking of growth factor receptors. While all three mammalian Diaphanous-related (mDia1/2/3) formin GTPase effector proteins have been localized on endosomes, a role for their actin nucleation, filament elongation, and/or bundling remains poorly understood in the context of intracellular trafficking. In a study of a functional relationship between RhoB, a GTPase known to associate with both early- and late-endosomes, and the formin mDia2, we show that 1) RhoB and mDia2 interact on endosomes; 2) GTPase activity-the ability to hydrolyze GTP to GDP-is required for the ability of RhoB to govern endosome dynamics; and 3) the actin dynamics controlled by RhoB and mDia2 is necessary for vesicle trafficking. These studies further suggest that Rho GTPases significantly influence the activity of mDia family formins in driving cellular membrane remodeling through the regulation of actin dynamics.