Acute lung injury (ALI) is a condition of acute respiratory failure, characterized by diffuse pulmonary infiltrates and severe hypoxemia. During ALI, the acute phase of inflammation induces the recruitment of activated inflammatory cells, including macrophages and lymphocytes, to the damaged lesions. Nuclear factor (NF)-kappa B is a key protein in many signal transduction pathways, over-activation of which is followed by an approach of inflammation cells and release of pre-inflammation cytokines. The aim of the present study was to explore the effect of NF-kappa B P65 siRNA retroviruses on the activation of NF-kappa B signaling pathway and release of pro-inflammatory cytokines in THP-1 cells. In the present study, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting were used to detect the NF-kappa B p65 mRNA and protein expression at different times in THP-1 cells infected by p65 siRNA retroviruses. The results revealed that p65 siRNA retroviruses could significantly inhibit the expression levels of NF-kappa B p65 mRNA and protein at different times. In addition, to further investigate the effect of p65 siRNA retroviruses on the pro-inflammatory cytokines release stimulated by LPS, the expression of IL-1 ss in THP-1 cells and TNF-alpha in THP-1/M cells was also detected using RT-qPCR and ELISA. As a result, the level of released proinflammatory cytokine interleukin-1 ss and tumor necrosis factor-alpha stimulated was significantly inhibited at different times infected by p65 siRNA retroviruses, while increased at different times infected by siControl retroviruses in THP-1 and THP-1/M cells stimulated by LPS. In summary, the present study demonstrated that p65 siRNA retroviruses could suppress the activation of NF-kappa B signal pathway and release of pro-inflammatory cytokines in THP-1 cells which provided a clinically plausible method to inhibit the inflammation for ALI/ARDS utilizing RNA interference technology.