Caspase-2 has been implicated in apoptosis and in non-apoptotic processes such as cell cycle regulation, tumor suppression and ageing. Using caspase-2 knockout (casp2−/−) mice, we show here that the putative anti-ageing role of this caspase is due in part to its involvement in the stress response pathway. The old casp2−/− mice show increased cellular levels of oxidized proteins, lipid peroxides and DNA damage, suggesting enhanced oxidative stress. Furthermore, murine embryonic fibroblasts from casp2−/− mice showed increased reactive oxygen species generation when challenged with pro-oxidants. Reduced activities of antioxidant enzymes glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were observed in the old casp2−/− mice. Interestingly, in the old casp2−/− animals expression of FoxO1 and FoxO3a was significantly reduced, whereas p21 levels and the number of senescent hepatocytes were elevated. In contrast to young wild-type mice, the casp2−/− animals fed an on ethanol-based diet failed to show enhanced GSH-Px and SOD activities. Thus, caspase-2, most likely via FoxO transcription factors, regulates the oxidative stress response in vivo.