Objective To explore the expression of myeloid differentiation protein2 ( MD-2) in rat lung and its role in acute lung injury ( ALI) induced by lipopolysaccharide ( LPS) . Methods Twenty male SD rats were randomly divided into a LPS group and a control group. The wet/dry ratios of lung tissues were measured and the histological changes of lung tissues were observed under microscope. Alveolar macrophages were collected from bronchial alveolar lavage fluid ( BALF) . The MD-2 mRNA and protein expressions were detected by RT-PCR, Western blot, and immunohistochemistry respectively. The MD2-siRNA oligo were transfected into NR8383 cells and 1 μg/mL LPS was used to stimulate the cells. The expressions of MD-2 mRNA and protein were detected by RT-PCR and Western blot. The levels of TNF-αin rat serum and cell culture supernatant were detected by ELISA. Results Compared with the control group, the expressions of MD-2 mRNA and protein in alveolar macrophages and lung tissue were elevated ( P lt;0. 01) , as well as the level of TNF-αin rat serum. The expressions of MD-2 mRNA and protein in NR8383 cell and the level ofTNF-αin supernatant increased obviously after LPS stimulation ( P lt;0. 01) . There were no changes of MD-2 mRNA and protein expressions and TNF-α of NR8383 cells treated by MD-2 siRNA with or without LPS stimulation ( P gt;0. 05) . Conclusions The expression of MD-2 in lung increases obviously after challengedby LPS. KnockdownMD-2 gene of NR8383 cell byMD-2 siRNA can inhibit TNF-αsecretion induced by LPS stimulation.MD-2 may play an important role in rat ALI induced by LPS.