Objective To observe the mechanism of the regulation of TLR9/MyD88 signaling pathway by Sanjulihe Prescription in the treatment of type Ⅲ prostatitis. Methods Establish the type Ⅲ prostatitis model rats and then randomly divided them into blank group, sham-operated group, model-control group, control drug group, and low, medium, and high dose groups of Sanjulihe Prescription, and were given the appropriate drug interventions for 4 consecutive weeks. At the end of drug administration, the animals were executed to obtain prostate tissues, which were subjected to HE staining to observe pathological changes, Elisa method to detect serum inflammatory factor indexes, Western blot to detect the expression level of genes and proteins related to TLR9/MyD88 signaling pathway, and PCR method to detect the expression level of genes related to TLR9/MyD88 signaling pathway. Results Sanjulihe Prescription can effectively improve the pathological changes of prostate tissue in type Ⅲ prostatitis model rats, and can improve inflammatory cell infiltration. Sanjulihe Prescription could reduce the levels of serum-related inflammatory factors IL-6, IL-17, and increase the level of regulatory cytokine IL-10 in model rats, and the efficacy of the drug showed a positive correlation with the dose. It can also down-regulate the expression of TLR9/MyD88 signaling pathway related genes TLR9, MyD88, IRAK1, IRAK4, TRAF6 proteins and genes, and there is a certain quantitative relationship between the efficacy of the drug and the dose, and the best effect is achieved when using a high dose of Sanjulihe Prescription. Conclusion From the experimental results, it was hypothesized that the treatment of type III prostatitis by Sanjulihe Prescription might be related to its modulation of TLR9/MyD88 signaling pathway.