The selenocysteine tRNA (tRNA^Sec) molecule is the sight of synthesis for the amino acid selenocysteine and the adaptor for its translational insertion into selenoprotein enzymes, the majority of which contribute to cellular redox homeostasis. To examine the consequences of selenoprotein depletion on the oxidative environment of the cell, we generated a conditional knockout mouse for the tRNA^Sec gene (Trsp). Deletion of Trsp, in either macrophage or liver, elevated oxidative stress and activated the transcriptional induction of cytoprotective antioxidant and detoxification enzyme genes including glutathione S-transferases P1 and NAD(P)H quinone oxidoreductase 1, and other well known target genes of the transcription factor Nrf2 (NF-E2 related factor 2). Simultaneous disruption of Trsp and Nrf2 severely compromised the cytoprotective response. Double knockout macrophages displayed a reduced viability, elevated oxidative stress, and an increased susceptible to hydrogen peroxide treatment over deletion of either gene alone. Mice carrying a liver specific deletion of Trsp on an Nrf2-null background experienced hepatocellular apoptosis and displayed a severely reduced survival rate over loss of Trsp alone. Our results thus demonstrate that reduced selenoprotein activity is counterbalanced by an Nrf2-mediated cytoprotective response, which is essential for maintaining cellular redox homeostasis and viability.