The aim of this study was to determine the functional significance of peroxiredoxin V (PRXV) in defense against oxidative stress and changes of its expression in human lung inflammation. We used in vitro cell cultures and retrospective analyses of human sputum samples to perform the study. We found that stable clones of lung epithelial cell lines A549 and U1810 with reduced expression of PRXV were prone to oxidative damage. Upregulation of PRXV decreased induction of DNA double-strand breaks and protein oxidation by cigarette smoke extract and hydrogen peroxide. Transfection with PRXV-carrying plasmid protected Calu-3 confluent epithelial cell sheets from alterations in barrier permeability induced by oxidative stress. In human sputum proinflammatory cytokines, myeloperoxidase, and PRXV were increased during viral-induced inflammation. We conclude that PRXV is an important antioxidant protein of lung epithelial cells. Its expression in the human lung increases in inflammation.