Nephrin is a slit diaphragm protein critical for structural and functional integrity of visceral glomerular epithelial cells (podocytes) and is known to be tyrosine phosphorylated by Src family kinases. We studied the role of phosphoinositide 3-kinase (PI3K), activated via the phosphorylation of nephrin, in actin cytoskeletal reorganization of cultured rat podocytes. Phosphorylation of rat nephrin by the Fyn kinase markedly increased its interaction with a regulatory subunit of PI3K. Stable transfection of rat nephrin in the podocytes with podocin led to nephrin tyrosine phosphorylation, PI3K-dependent phosphorylation of Akt, increased Rac1 activity, and an altered actin cytoskeleton with decreased stress fibers and increased lamellipodia. These changes were reversed with an inhibitor of PI3K and not seen when the nephrin-mutant Y1152F replaced wild-type nephrin. Rac1 and Akt1 contributed to lamellipodia formation and decreased stress fibers, respectively. Finally, in the rat model of puromycin aminonucleoside nephrosis, nephrin tyrosine phosphorylation, nephrin-PI3K association, and glomerular Akt phosphorylation were all decreased. Our results suggest that PI3K is involved in nephrin-mediated actin reorganization in podocytes. Disturbed nephrin-PI3K interactions may contribute to abnormal podocyte morphology and proteinuria.