The pathogenesis of acute lung injury (ALI) is characterized by lung inflammation and lung oxidative stress. The study was conducted in order to investigate the effect toll-like receptor 4 (TLR4) and nuclear factor-kappa B (NF-kappa B) exhibited on oxidative stress in ALI. After the rats had been assigned into different groups, arterial blood, white blood cell (WBC), lung permeability index (LPI), wet/dry (W/D) ratio, TLR4 and NF-kappa B expression and superoxide dismutase (SOD), myeloperoxidase (MPO), malondialdehyde (MDA), glutathione (GSH), and reactive oxygen species (ROS) were examined. Afterward, the correlation between the levels of TLR4 and NF-kappa B was determined. Decreased levels of PaO2, SOD, MPO, and GSH accompanied by increased levels of PaCO2, WBC number, LPI and W/D ratio, MDA and ROS, as well as TLR4 and NF-kappa B expressions in the ALI, ALI+NF-kappa B inhibitor, and ALI + phosphate buffer saline groups were found. Inhibition of NF-kappa B resulted in increased PaO2 and decreased PaCO2 levels, WBC number, and LPI and W/D ratio. Decreased expression of NF-kappa B increased SOD, GSH, and MPO, but decreased MDA and ROS. We also found that NF-kappa B inhibition resulted in the improvement of ALI in rats. TLR4 and NF-kappa B expressions were negatively correlated with levels of SOD, MPO, and GSH, and positively correlated with MDA and ROS levels. In summary, our findings provided evidence that inhibition of the TLR4/NF-kappa B signaling pathway decreases oxidative stress, thereby improving ALI. As a result, NF-kappa B signaling pathway has shown potential as a therapeutic target in ALI therapy.