High-tidal-volume mechanical ventilation and hyperoxia used in patients with acute lung injury (ALI) can induce alveolar coagulopathy and fibrin depositions within the airways. Hyperoxia has been shown to increase ventilator-induced lung injury (VILI), but the mechanisms that regulate interaction between high-tidal-volume mechanical ventilation and hyperoxia are unclear. We hypothesized that mechanical stretch with hyperoxia synergistically augmented neutrophil infiltration and production of plasminogen activator inhibitor-1 (PAI-1) via the nuclear factor-κB (NF-κB) pathway. C57BL/6 mice (n = 5 per group) were exposed to high-tidal-volume (30mL/kg) or low-tidal-volume (6mL/kg) mechanical ventilation with room air or hyperoxia for 1 to 5h after 2-μg/g NF-κB inhibitor (SN-50) administration. Nonventilated mice with room air or hyperoxia served as control groups. Evans blue dye, myeloperoxidase, electrophoretic mobility shifting of nuclear protein, and inflammatory cytokine were measured. The expression of tumor necrosis factor-α (TNF-α) and PAI-1 were studied by immunohistochemistry. The addition of hyperoxia to high-tidal-volume ventilation-augmented lung injury, as demonstrated by increased microvascular leak, neutrophil migration into the lung, TNF-α and active PAI-1 production, DNA binding activity of NF-κB, and NF-κB activation. No statistically significant increase of neutrophil infiltration and inflammatory cytokine production was found in the mice ventilated at 6mL/kg using hyperoxia. Hyperoxia-induced augmentation of VILI was attenuated in mice with pharmacologic inhibition of NF-κB activity by SN-50. We conclude that hyperoxia increased high-tidal-volume-induced cytokine production and neutrophil influx through activation of the NF-κB pathway.