NRF2 is a transcription factor important in the protection against carcinogenesis and oxidative stress through antioxidant response element (ARE)-mediated transcriptional activation of several phase 2 detoxifying and antioxidant enzymes. This study was designed to determine the role of NRF2 in the pathogenesis of hyperoxic lung injury by comparing pulmonary responses to 95–98% oxygen between mice with site-directed mutation of the gene for NRF2 (Nrf2 ^{− / −}) and wild-type mice (Nrf2 ^{+ / +}). Pulmonary hyperpermeability, macrophage inflammation, and epithelial injury in Nrf2 ^{− / −} mice were 7.6-fold, 47%, and 43% greater, respectively, compared with Nrf2 ^{+ / +} mice after 72 h hyperoxia exposure. Hyperoxia markedly elevated the expression of NRF2 mRNA and DNA-binding activity of NRF2 in the lungs of Nrf2 ^{+ / +} mice. mRNA expression for ARE- responsive lung antioxidant and phase 2 enzymes was evaluated in both genotypes of mice to identify potential downstream molecular mechanisms of NRF2 in hyperoxic lung responses. Hyperoxia-induced mRNA levels of NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione-S-transferase (GST)-Ya and -Yc subunits, UDP glycosyl transferase (UGT), glutathione peroxidase-2 (GPx2), and heme oxygenase-1 (HO-1) were significantly lower in Nrf2 ^{− / −} mice compared with Nrf2 ^{+ / +} mice. Consistent with differential mRNA expression, NQO1 and total GST activities were significantly lower in Nrf2 ^{− / −} mice compared with Nrf2 ^{+ / +} mice after hyperoxia. Results demonstrated that NRF2 has a significant protective role against pulmonary hyperoxic injury in mice, possibly through transcriptional activation of lung antioxidant defense enzymes.