Wilms tumor interacting protein (Wtip) has been implicated in cell junction assembly and cell differentiation and interacts with proteins in the podocyte slit diaphragm, where it regulates podocyte phenotype. To define the Wtip expression and function in the kidney, we created a Wtip-deleted mouse model using the β-galactosidase-neomycinR (β-geo) gene trap technology. Wtip gene trap mice were embryonic lethal, suggesting additional developmental roles outside kidney function. Using β-geo heterozygous and normal mice, Wtip expression was identified in developing kidneys, heart, and eyes. In the kidney, expression was restricted to podocytes, which appeared initially at the capillary loop stage coinciding with terminal podocyte differentiation. Heterozygous mice had an expected lifespan and showed no evidence of proteinuria or glomerular pathology. However, heterozygous mice were more susceptible to glomerular injury than wild-type littermates and developed more significant and prolonged proteinuria in response to LPS or adriamycin. In normal human kidneys, WTIP expression patterns were consistent with observations in mice and were lost in glomeruli concurrent with loss of synaptopodin expression in disease. Mechanistically, we identified the Rho GTPase guanine nucleotide exchange factor ARHGEF12 as a binding partner for WTIP. ARHGEF12 was expressed in human podocytes and formed high-affinity interactions through their LIM and PDZ binding domains. Our findings suggest that Wtip is essential for early murine embryonic development and maintaining the normal glomerular filtration barrier function, potentially regulating slit diaphragm and foot process function through Rho effector proteins.