P66Shc knockout mice exhibit increased longevity due to their resistance to oxidative stress-induced DNA damage, apoptosis and disease. Herein this study we examined the mechanistic role of p66Shc in controlling the oxidative stress response of early preimplantation stage embryos. Utilizing RNA interference -mediated knockdown of p66Shc in bovine zygotes, we demonstrate that p66Shc regulates intracellular ROS-induced DNA damage that leads to permanent embryo arrest and apoptosis. P66Shc knockdown embryos are stress resistant exhibiting reduced intracellular ROS levels, during in vitro embryo development. The aim of this study was to elucidate the functional role of p66Shc throughout early embryo development. We have previously determined that elevated p66Shc mRNA and protein abundance in early embryos are associated with reduced developmental potential. We have also shown that the early embryo’s response to reactive oxygen species is developmental regulated and that activated p66Shc is linked to increased intracellular ROS levels, reduced antioxidant expression and to permanent embryo arrest. Previous microinjections of short hairpin RNAi molecules targeting p66Shc in immature bovine oocytes successfully knockdown p66Shc and alleviated the 2–4 cell block observed after fertilization, but also resulted in reduced blastocyst development suggesting a role for p66Shc beyond early cleavage arrest. Herein, we mechanistically demonstrate for the first time that RNAi-mediated knockdown of p66Shc at the zygote stage confers oxidative stress resistance throughout early bovine embryo development. P66Shc knockdown embryos are more resistant to oxidant treatment due to increased Perifosine side effects levels of MnSOD and a reduction in intracellular ROS and DNA damage content that diminishes the frequencies of both permanent embryo arrest and apoptosis resulting in improved blastocyst development in vitro. According to a series of criteria, two non-overlapping siRNA sequences were designed to specifically target the unique CH2 domain of bovine p66Shc. These siRNAs were different than the previous p66Shc shRNA sequence utilized in our previous study. The use of multiple target sequences of the same RNA molecule is an effective means of verifying a genuine loss-of-function effect. While both siRNA molecules induced a significant decrease in p66Shc mRNA and protein, RNAi-E was the more effective of the two. The accessibility to the target sequence may be greater for RNA-E molecules than for RNAi-A siRNAs. A difference in the internal stability of the two siRNA molecules could manifest as different rates of strand selection and entry into the RNA-induced silencing complex leading to differential knockdown efficiencies. A significant increase in p66Shc mRNA and protein was observed in embryos injected with negative control siRNAs compared to noninjected embryos. The microinjection process, which involves prolonged exposure to ambient temperature and oxygen tensions, as well as disruption of the cell membrane and components of the cytoskeleton, is a significant stress upon the early bovine embryo. This upregulation may mask the true knockdown effect of p66Shc siRNA molecules, which may prove more effective if introduced in a less traumatic manner. Attempts introducing siRNAs into bovine zygotes using lipofectamine were futile.