The aim of the present study was to investigate the effects of Wumei Pill on the TLR4/9-NF-kB signaling pathway in rats with diarrhea-predominant irritable bowel syndrome (IBS-D). The model was established by means of acetic acid enema plus restraint stress. The rat fecal moisture content, fecal trait score, and abdominal wall withdrawal reflex (AWR) score were used to determine whether the model was successful. The effects of Wumei Pill on the TLR4/9-NF-kB signaling pathway rats with IBS-D was observed by testing the contents of serum level of tumour necrosis factor-α (TNF-a), interleukin-6 (IL-6), lipopolysaccharide (LPS) as well as colonic mucosal tissue TLR4/9 and NF-kB. We got the following results: (1) The rat IBS-D model was successfully prepared; (2) Wumei Pill could reduce the contents of serum TNF-α, IL-6 and LPS in rats(P<0.05), and alleviate the inflammatory reaction and accumulation of bacterial endotoxins; (3) Wumei Pill could reduce the transcriptional level of TLR4/9mRNA, to decrease TLR4/9 protein synthesis and expression. So we concluded that Wumei Pill probably reduce the release of pro-inflammatory cytokines and the bacterial endotoxins by blocking the transmission of TLR4/9-NF-kB signaling pathway so that Wumei Pill has a significant therapeutic effect on IBS-D.