In glomerular disease, podocyte injury results in a dramatic change in cellmorphology known as foot process effacement. Remodeling of the actin cytoskeleton through the activity of small GTPases was identified as a key mechanism in effacement, with increased membrane activity and motility in vitro. However, whether podocytes are stationary or actively moving cells in vivo remains debated. Using intravital and kidney slice two-photon imaging of the three-dimensional structure of mouse podocytes, we found that uninjured podocytes remained nonmotile andmaintained a canopy-shaped structure over time. On expression of constitutively active Rac1, however, podocytes changed shape by retracting processes and clearly exhibited domains of increased membrane activity. Constitutive activation of Rac1 also led to podocyte detachment from the glomerular basement membrane, and we detected detached podocytes crawling on the surface of the tubular epithelium and occasionally, in contact with peritubular capillaries. Podocytemembrane activity also increased in the inflammatory environment of immune complex-mediated GN. Our results provide evidence that podocytes transition from a static to a dynamic state in vivo, shedding new light on mechanisms in foot process effacement.