Objective To construct the replication-incompetent lentivirus expressing rat TLR4 shRNA, and to observe its inhibitory effect on TLR4 expression in alveolar macrophage and on lipopolysaccharide (LPS)-induced release of IL-1β, IL-6. Methods We designed and constructed four shRNA expressing plasmids with silencing effect, then they were co-transfected into HEK-293T cells with the TLR4 eukaryotic vector constructed previously; the best shRNA was selected to be packed into lentivirus; and the titer was determined. The packed lentvirus was used to infect the alveolar macrophage(NR8383) in presence of LPS, and the LPS-induced IL-1β, IL-6 expression was examined by ELISA. Results The best shRNA was successfully screened out and was correctly inserted into the lentivirus. The titer of the recombinant lentivirus was 2.0×106TU/ml. The LPS-induced expression of IL-1β, IL-6 in the alveolar macrophages was greatly reduced after virus infection(P<0.05). Conclusion We have successfully constructed the recombinant lentivirus expressing rat TLR4 shRNA, which has an satisfactory inhibitory effect against expression of IL-1β, IL-6, paving a way for studying the chronic rejection reaction after rat lung transplantion in vivo.