The Free Radical Theory of Aging proposes that reactive oxygen species (ROS) contribute to the pathophysiology of aging. Our previous data highlight the importance of antioxidant enzymes, superoxide dismutase 1 (Sod1) and glutathione peroxidase 1 (Gpx1), in regulating this process. Previously, we demonstrated that a perturbation in the Sod1-to-Gpx1 ratio, as a consequence of Sod1 overexpression, leads to senescence-like changes. We proposed that this was mediated via the Sod1 dismutation product H₂O₂, because H₂O₂ induced similar changes in control cells. However, it has been suggested that H₂O₂ production, via Sod1 dismutation, is rate-limited by the availability of the substrate O₂⁻, and therefore age-related changes may occur as a result of other functions of Sod1. In this study, we test this notion in fibroblasts derived from Gpx1 null mutant mice (Gpx1−/−) that have elevated H₂O₂ as a consequence of the lack of its removal by Gpx1. We demonstrate senescence-like changes in Gpx1−/− fibroblasts that include (1) reduced proliferative capacity, DNA synthesis, and responsiveness to EGF and serum; (2) elevated levels of Cip1; (3) increased NF-κB activation; and (4) morphological features of senescent cells. Gpx1−/− fibroblasts also demonstrate a dose-dependent susceptibility to H₂O₂-induced apoptosis. Our findings suggest that Gpx1 is protective against both ROS-mediated senescence-like changes and oxidant-mediated cell death.