Objective To investigate the effects of interferon regulator factor 3 (IRF3) shRNA on the expression of TLR4 downstream signal molecules including IRF3-IFN-β, NF-κB/p38 MAPK-TNF-α/IL-1β and IL-10 in lipopolysaccharide (LPS)-stimulated Kupffer cells (KCs). Methods KCs were isolated from rats by in situ perfusion. The adenovirus strains carrying IRF3 shRNA were used for the transfection of purified KCs. The isolated KCs were randomly divided into four groups including adenovirus(-) LPS(-) treatment group, adenovirus(-) LPS(+ ) treatment group, adenovirus(+ ) LPS(-) treatment group and adenovirus(+ ) LPS(+ ) treatment group. The levels of cytokines in the supernatants of KC culture were detected by enzyme-linked immunosorbent assay (ELISA). Real-time PCR and Western blot assay were performed to analyze the expression of related cytokines at mRNA and protein levels, respectively. Results The expression of IRF3 at mRNA and protein levels in primary cultured KCs were induced by LPS. The cellular constitutive expression of IRF3 at mRNA level and the LPS-induced expression of IRF3 were significantly inhibited after transfection of KCs with adenovirus strains carrying IRF3 shRNA. However, the nuclear constitutive expression of IRF3 protein was not affected by IRF3 shRNA. The expression of IFN-β at mRNA and protein levels in KCs were induced by LPS, but were suppressed by the interference with IRF3 shRNA. No significant changes of the cellular constitutive expression of IFN-β at mRNA and protein levels were observed in IRF3 shRNA-treated KCs. Enhanced expression of proinflammatory cytokines including TNF-α and IL-1β at mRNA and protein levels were detected in LPS-stimulated KCs. Transfection of KCs with adenovirus strains carrying IRF3 shRNA inhibited the LPS-induced secretion of TNF-α and IL-1β, but neither LPS-induced expression of TNF-α and IL-1β at mRNA level nor cellular constitutive expression of TNF-α and IL-1β at mRNA and protein levels were affected by IRF3 shRNA. The LPS-induced expression of IL-10 at mRNA and protein levels were enhanced in IRF3 shRNA-treated KCs. However, the cellular constitutive expression of IL-10 at mRNA and protein levels were not affected by the adenovirus. The levels of phosphor-NF-κB p65 subunit and phosphor-p38 MAPK protein in the nuclei of KCs were increased upon the stimulation with LPS. Treatment of KCs with IRF3 shRNA showed no significant effects on nuclear phosphor-NF-κB p65 subunit and phosphor-p38 MAPK. Conclusion Transfection of LPS-stimulated primary KCs with adenovirus strains carrying IRF3 shRNA could effectively inhibit the expression of IRF3 and the transduction of downstream signals. IRF3 enhanced the secretion of TNF-α and IL-1β, but inhibited the expression of IL-10 in LPS-treated KCs. The LPS-induced activation of NF-κB and p38 MAPK in KCs were not affected by IRF3 signal. Key words: IRF3; shRNA adenovirus; Signal pathway; LPS; Primary Kupffer cells