Objective To approach the actions of nitric oxide (NO) and inducible nitric oxide synthase (iNOS) on endotoxin (LPS) induced rat acute lung injury (ALI), and the affection of Rhubarb on them. Methods LPS was injected into the sublingual vein of male Wistar rats to prepare ALI animal models. 160 rats were divided into 4 groups: LPS group, control group, Rhubarb treatment group and dexamethasone group. Histopathological examinations were performed and biological indexes were measured for the lung specimens. The indexes included wet/dry weight, the rate of neutrophils and protein content in the pulmonary alveolar lavage fluid, pulmonary vascular permeability and pulmonary alveolar permeability index. The activities of serum NO and lung tissue iNOS were measured. Results In the LPS group, the lung wet/dry weight (4.47± 0.14 vs 4.18 ± 0.28), the rate of neutrophils (49.21%± 8.52% vs 23.29% ± 9.84%), protein content (240.33± 109.67 μg/mg vs 62.17 ± 19.13 μg/mg) were significantly higher than those in the control group (P <0.01). In the Rhubarb and dexamethasone groups, the lung wet/dry weight (4.24 ± 0.14, 4.22 ± 0.16 vs 4.47 ±0.14), the rate of neutrophils (37.28 ± 8.72, 30.17 ±15.66 vs 49.21 ± 8.52), protein content (83.20 ± 45.6, 116.0 ± 38.47 vs 240.33 ± 109.67 μg/mg) were significantly lower than those in the LPS group (P < 0.05, P < 0.01) . The pulmonary alveolar permeability index and vascular permeability during ALI in LPS group (4.78 ±1.9, 6.08 ± 0.73 vs 1.90 ± 0.28, 2.7 ± 0.78 μg/ml, respectively) were significantly higher than those in the control group (P < 0.01) . After Rhubarb and dexamethasone treatment, the two indexes (R: 1.59 ±0.52, 3.42 ± 1.25; D: 2.88 ± 0.19, 3.9±0.78 μg/ml) were significantly lower than those in the LPS group (P <0.05, P < 0.01). The concentrations of serum NO and lung tissue iNOS in the LPS group were significantly higher than those in the control group (NO: 3.12 ± 0.88 vs 2.10 ±0.34 μg/ml; iNOS: 109.87 ± 50.28 vs 37.25 ± 12.1, respectively. P < 0.05, P < 0.01). After Rhubarb and dexamethasone treatments, the serum NO level (R: 1.65 ±0.97 μg/ml; D: 1.26 ± 0.69 μg/ml) and the concentration of iNOS (R: 49.50 ± 13.46; D: 40.77 ± 6.91) in the lung tissue in the ALI were significantly lower than those in the LPS group (P <0.05, P <0.01). Under light microscope, in the LPS group, there were diffuse hemorrhage in the lung stroma and a large amount accumulate of erythrocytes and neutrophils in the alveolar cavities, the lung presented widespread congestion, edema, alveolar septum thickness, fluid accumulation in alveolar cavity. But in the Rhubarb and dexamethasone group, these changes in ALI were drastically ameliorative. Conclusion From the LPS induced ALI rat models, it is demonstrated that NO and iNOS play important roles in the onset of ALI. Dexamethasone and Rhubarb treatment can ameliorate the lung injury and decrease the concentrations of NO and iNOS, showing that these two agents have protective effect on LPS induced ALI and their therapeutic mechanism is possibly through inhibiting the activities of NO and iNOS.