MAGUK with inverted domain structure-1 (MAGI-1) is a membrane-associated protein with one guanylate kinase, six PSD-95/Dlg-A/ZO-1 (PDZ), and two WW domains and is localized at tight junctions in epithelial cells. MAGI-1 interacts with various proteins and is proposed to function as a scaffold protein. In the previous study, we discovered a MAGI-1-interacting cell adhesion molecule junctional adhesion molecule 4 (JAM4). Both proteins are highly expressed in glomerular podocytes in the kidney and partially colocalized. In this study, we have further searched for a binding partner of MAGI-1 in the kidney through yeast two-hybrid screening and obtained nephrin. Nephrin is a cell adhesion molecule specifically localized at the slit diaphragm between neighboring foot processes of podocytes. Biochemical studies reveal that nephrin directly binds to the middle PDZ domains of MAGI-1 through its carboxyl terminus but does not bind to ZO-1. MAGI-1 forms a tripartite complex with nephrin and JAM4 in vitro. Immunoelectron microscopy shows that the localization of MAGI-1 is restricted to the slit diaphragm, whereas JAM4 is also distributed on apical membranes of podocytes. In puromycin aminonucleoside-induced nephrotic podocytes, MAGI-1 is localized with nephrin at the displaced slit diaphragm. These data indicate that MAGI-1 is a component of the slit diaphragm and tightly interacts with nephrin and JAM4 in vivo. MAGI-1 may play a role in determining the boundary between the apical and the bosolateral domain at the level of slit diaphragm.