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 knock-out mouse for the tRNA(Sec) gene (Trsp). Deletion of Trsp in either macrophages or liver elevated oxidative stress and activated the transcriptional induction of cytoprotective antioxidant and detoxification enzyme genes, including glutathione S-transferase 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 knock-out macrophages displayed reduced viability, elevated oxidative stress, and increased susceptible to hydrogen peroxide treatment compared with 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 compared with 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.