The slit diaphragm and the apical and basal membrane domains of podocytes are connected to each other by an actin-based cytoskeleton critical to the maintenance of the glomerular filtration barrier. In an effort to discover novel regulatory proteins of the podocyte foot process, we identified and characterized pdlim2, a member of the actin-associated LIM protein subfamily of cytosolic proteins typified by an N-terminal PDZ domain and a C-terminal LIM domain. In the kidney, the pdlim2 protein is highly specific for the glomerulus and podocyte foot processes as shown by RT-PCR, western blotting, immunofluorescence, and immunoelectron microscopy. In cultured podocytes, pdlim2 was associated with stress fibers and cortical actin. Pdlim2 seems to regulate actin dynamics in podocytes since stress fibers were stabilized in its presence. Mechanistically, pdlim2 interacts with two actin-associated podocyte proteins, α-actinin-4 and angiomotin-like-1, as shown by immunoprecipitation and yeast two-hybrid analyses. By semi-quantitative immunoelectron microscopy, there was a reduced expression of pdlim2 in podocytes of patients with minimal change nephrotic syndrome and membranous nephropathy, whereas its expression was unchanged in patients with focal segmental glomerulosclerosis. Hence, pdlim2 is a novel actin-regulating protein of podocyte foot processes that may have a role in the pathogenesis of glomerular diseases.