Objective To study the effect of hemofiltration plus hemoperfusion (HF+HP) on removal of serum TNF-α in severe acute pancreatitis (SAP) patients with lung injury and on RhoA serine 188 phosphorylation (p-RhoA) in endothelial cells. Methods Totally 35 SAP patients, including 28 with acute lung injury (ALI)/ acute respiratory distress syndrome (ARDS), and 20 healthy subjects were involved in this study. All the SAP patients were treated in ICU, and the 9 combined with ARDS underwent continuous veno-venous hemofiltration (CVVH) and HP therapy (HF+HP group). The serum TNF-α in SAP patients with different degrees of lung injury and the serum TNF-α in SAP patients with ARDS was determined by ELISA at different time points during HF+HP. For in vitro study the human umbilical vein endothelial cells (HUVECs) were treated with sera from SAP patients with ARDS during HF+HP, and those treated with recombinant TNF-α were taken as a positive control. The changes of p-RhoA and total RhoA were observed by Western blotting analysis. Subcellular distribution of p-RhoA protein was observed by immunofluorescence staining. Results We found that, compared with SAP patients without ALI/ARDS, those with ALI/ARDS, especially those with ARDS (about 7 folds that of the normal control) had a significantly increased serum TNF-α level (P<0.05). The serum TNF-α level began to decrease in the 9 SAP patients with ARDS 6 h after HF+HP treatment, with the decrease reached maximal 20 h after treatment, closing to the level of the normal control group. Meanwhile, the arterial blood PaO₂ , HCO3- and PaO₂/FiO₂ ratio were significantly elevated after HF+HP treatment in SAP patients with ARDS(P<0.05). Western blotting analysis showed that p-RhoA level was significantly increased in HUVECs treated with the serum of HF+HP-treated SAP patients with ARDS (P<0.05), and the total RhoA protein expressions were not significantly different among different groups (P>0.05). Immunofluorescence indicated that p-RhoA was largely distributed in the cytoplasm of HUVECs, and the changes were consistent with the data from Western blotting analysis. Conclusion SAP patients with ALI/ARDS have high circulation TNF-α. Treatment with HF+HP can effectively remove the excessive TNF-α in the blood of SAP patients with ALI/ARDS and inhibit activation of RhoA, subsequently decreasing the high permeability of endothelial cells.