The regulation of Rho GTPase activities and expression is critical in the development and function of the kidney. Rho GTPase activities and cytosol-membrane cycling are regulated by Rho GDP Dissociation Inhibitor (RhoGDI), and RhoGDI knockout mice develop defects in kidney structure and function that lead to death due to renal failure. It is therefore important to understand the changes in RhoGDI-regulated Rho GTPase activities and cell morphology that lead to kidney failure in RhoGDI (-/-) mice. Here, we characterize a renal mesangial cell line derived from the RhoGDI (-/-) mouse in which we verify the absence of GDI proteins. In the absence of RhoGDI, we show an increase in the specific activity of Rac1, and to a lesser extent, RhoA and Cdc42 GTPases in these cells. This is accompanied by a compensatory decrease in the steady-state protein levels of Rho GTPases. Morphological analysis of RhoGDI (-/-) mesangial cells reveals a decrease in cell spreading and in focal contacts compared to wild-type cells. Finally, RhoGDI (-/-) mesangial cells show a decreased ability to proliferate and survive. These functional and structural changes are likely to contribute to the defects in renal architecture and function observed in the RhoGDI (-/-) mouse.