This paper aims to explore the mechanism of Qianlie-Biertong suppository (QS) in improving prostatitis in vitro.Methods:The intestine absorbed drug solution of QS (IQS) was prepared ex vivo and characterized by UPLC-QE-Orbitrap-MS.The lipopolysaccharide (LPS)-induced PC-3 cell inflammation model of human prostate cancer cell lines was established.Cell viability was determined by MTT assay.The gene expression levels of inflammatory mediators were detected by qRT-PCR.The expression levels of the key proteins involved in the Toll-like receptor 4 (TLR4) signaling pathway were determined by Western blot.The nuclear translocation of nuclear factor κB/p65，activated protein-1 (AP-1) subunit c-Jun，and interferon regulatory factor 3 (IRF3) was detected by immunofluorescence.Results:Various bioactive components including berberine，chlorogenic acid，and ferulic acid were identified in IQS.IQS treatment at concentrations of 6.25 to 400 μg/mL had no significant effect on the cell viability of PC-3 cells.The gene expression levels of inflammatory mediators under LPS exposure，including TNFA，IL-6，CXCL10，MCP1，and COX2 were markedly inhibited by IQS.IQS could downregulate the expression levels of phosphorylated AKT，IκBα，IKKα/β，p65，p38，c-Jun，TBK1，and IRF3 in a dose-dependent manner，which were key proteins of the TLR4 signaling pathway.Furthermore，IQS remarkably suppressed the nuclear translocation of p65，c-Jun，and IRF3 induced by LPS.Conclusion:IQS mitigated LPS-induced inflammatory response in PC-3 cells，which may be associated with the inhibitory effects of IQS on the TLR4 signaling pathway.