Supplemental oxygen (O₂) increases the risk of lung injury in preterm infants owing to an immature antioxidant system. Our objective was to determine whether impairing antioxidant defense by decreasing glutathione peroxidase 1 (GPx1) gene expression increases the injurious effects of hyperoxia. GPx1^+/+ and GPx1^-/- C57Bl/6J mice were exposed to 21% O₂ (Air) or 40% O₂ (Hyperoxia; Hyp) from birth to postnatal day 7 (P7d); they were killed at P7d or maintained in air until adulthood (P56d) to assess short-term and long-term effects, respectively. We assessed lung architecture, three markers of pulmonary oxidative stress (P7d, P56d), macrophages in lung tissue (P7d), immune cells in bronchoalveolar lavage fluid (BALF) (P56d), and GPx1-4 and catalase gene expression in lung tissue (P7d, P56d). At P7d: macrophages were decreased by lack of GPx1 expression, and further decreased by hyperoxia. GPx1 expression was increased in GPx1^+/+Hyp mice and decreased in both GPx1^-/- groups. At P56d: heme oxygenase-1 was increased by hyperoxia when GPx1 was absent. There were significantly more immune cells from Hyp groups than from the GPx1^+/+Air group and greater proportion of lymphocytes in GPx1^-/-Hyp. GPx1 expression was significantly decreased in GPx1^-/- mice; GPx2-4 and catalase expression was increased in GPx1^-/-Hyp mice compared to other groups. Tissue fraction was decreased in GPx1^-/-Air mice; bronchiolar smooth muscle was decreased in GPx1^-/- mice. GPx1 does not clearly exacerbate hyperoxia-induced increases in oxidative stress or lung injury, but may alter pulmonary immune function. Increased expression of GPx2-4 and catalase in GPx1^-/-Hyp mice suggests gene redundancy within the model.