Slit3 along with Slit1 and Slit2 comprise the Slit family of proteins. The latter two proteins are known to be involved in axon guidance and cell migration during animal development. However, little is know about the functions of Slit3. We created a Slit3-deficient mouse model from an OmniBank ES cell line with a Slit3 allele trapped by insertional mutagenesis to analyze the in vivo functions of this protein. In this model, congenital diaphragmatic hernia is the most obvious phenotype. Herniation was found to be caused by a defective central tendon (CT) of the diaphragm that remained fused with the liver. Electron microscopic analyses of the defective CT revealed disorganized collagen fibrils that failed to form tight collagen bundles. The hearts of Slit3-deficient mice have an enlarged right ventricle. In addition, 20% of homozygous mice also showed a range of kidney defects that include unilateral or bilateral agenesis of the kidney and ureter, or varying degrees of renal hypoplasia. Thus, we concluded that Slit3 is involved in the development of multiple organ systems that include the diaphragm and the kidney. Slit3-deficient mice represent a genetic animal model for physiological and pathological studies of congenital diaphragmatic hernia.