Shc proteins, products of Shc1 gene, exists in three functionally distinct isoforms (p46Shc, p52Shc, and p66Shc) that serve as intracellular adaptors for several key signaling pathways involved in renal pathologies. Adaptor protein p66Shc contributes to the pathogenesis of oxidative stress-related diseases and is known to regulate the sensitivity to oxidative stress. The objective of this study was to test the hypothesis that abnormal p66Shc-mediated ROS production could be critically involved in the development of nephrons during nephrogenesis. p66Shc ability to promote oxidative stress-related diseases requires phosphorylation of serine 36 residue (Ser36), which causes conformation change and consequent translocation of p66Shc to the mitochondria where it increases the production of ROS. Using embryo microinjection of Zinc Finger Nucleases, we have generated unique mutant rats (termed p66Shcdel29-37), which express endogenous p66Shc with a 9-amino acid deletion containing the regulatory Ser36 on the genetic background of Dahl salt-sensitive (SS) rats. p66Shcdel29-37 rats display increased H2O2 renal production, as measured by enzymatic microelectrode biosensors when compared with parental SS rats. Since p66Shc is known to oxidize cytochrome c in mitochondria, rendering it unavailable to reduce oxygen to water and promoting deviation of a fraction of mitochondrial electron flow to the production of H2O2, likely p66Shcdel29-37 is constitutively translocated to mitochondria. The kidney is particularly sensitive to increases in oxidative stress, and p66Shcdel29-37 rats are characterized by increased susceptibility to renal pathologies and reduction in renal function. We quantitated nephron numbers in fully developed homozygote and heterozygote p66Shcdel29-37 rats as well as in the SS rats using the acid maceration method. We also quantified ureteric bud branching morphogenesis in embryos from corresponding strains using the ureteric bud marker calbindin-D-28K. Metanephric kidney ureteric bud branching is suppressed in embryos, and the number of glomeruli is significantly reduced in adults p66Shcdel29-37 rats when compared to SS. The mechanism of nephron number control is not well characterized. Our data suggest that H2O2 renal production caused by abnormal signaling of p66Shc could be critical in regulating nephrogenesis and constitutive p66Shc signaling negatively impacts kidney development and renal function.