Updated project metadata. Deleted in Liver Cancer 3 (DLC3) is a Rho GTPase-activating protein (RhoGAP) that plays a crucial role in maintaining adherens junction integrity and coordinating polarized vesicle transport by modulating Rho activity at the plasma membrane and endomembranes. By employing bioinformatical sequence analysis, in vitro experiments, and in cellulo assays a polybasic region (PBR) responsible for mediating the association of DLC3 with cellular membranes was identified. It was shown that the DLC3 PBR contains two novel phosphorylation sites that can alter the local electrostatic character of the PBR and its ability to interact with negatively charged membranes. Accordingly, upon introduction of phosphomimetic mutations into the DLC3 PBR, membrane association of DLC3 was impaired. Furthermore, we uncovered a distinct, PBR-dependent localization of DLC3 at the midbody during cell division, where it exerted control over local Rho activity.