α-Klotho exerts pleiotropic biological actions. Heterozygous α-Klotho haplo-insufficient mice (kl/+) appear normal at baseline except for age-related changes in the lung, suggesting heightened pulmonary susceptibility to α-Klotho deficiency. We used in vivo and in vitro models to test whether α-Klotho protects lung epithelia against injury. Normally, α-Klotho is not expressed in the lung, but circulating α-Klotho levels are reduced ∼40% in kl/+ mice and undetectable in homozygous α-Klotho-deficient mice (kl/kl). kl/+ mice show distal air space enlargement at a given airway pressure, with elevated lung oxidative damage marker (8-hydroxydeoxyguanosine; 8-OHdG); these abnormalities are exacerbated in kl/kl mice. Studies were performed in A549 lung epithelial cells and/or primary culture of alveolar epithelial cells. Hyperoxia (95% O₂) and high inorganic phosphate concentrations (Pi, 3–5 mM) additively caused cell injury (lactate dehydrogenase release), oxidative DNA damage (8-OHdG), lipid oxidation (8-isoprostane), protein oxidation (carbonyl), and apoptosis (caspase-8 activity and TUNEL stain). Transfection of transmembrane or soluble α-Klotho, or addition of soluble α-Klotho-containing conditioned media, increased cellular antioxidant capacity (Cu- and Fe-based assays) via increased nuclear factor erythroid-derived 2-related factors 1 and 2 (Nrf1/2) transcriptional activity and ameliorated hyperoxic and phosphotoxic injury. To validate the findings in vivo, we injected α-Klotho-containing conditioned media into rat peritoneum before and during hyperoxia exposure and found reduced alveolar interstitial edema and oxidative damage. We conclude that circulating α-Klotho protects the lung against oxidative damage and apoptosis partly via increasing endogenous antioxidative capacity in pulmonary epithelia. Cytoprotection by α-Klotho may play an important role in degenerative diseases of the lung.