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Background It is urgently necessary to find new drugs for chronic prostatitis (CP) as desired treatment effect is still yet to be achieved. Our previous clinical study has found that Anemone tomentosa (Maxim.) Péi has a good effect on CP, but the specific mechanism of action remains to be further studied. Objective To investigate the role of prostasome-derived miRNA-146a in regulating toll-like receptor 4/nuclear factor-κB (TLR-4/NF-κB) pathway and the mechanism of action of Anemone tomentosa in treating chronic inflammation in experimental autoimmune prostatitis (EAP) rats. Methods This study was conducted between December 2021 and June 2022. Forty-two Wistar rats were equally divided into seven groups using a random number table, including normal group, model group, low-, medium- and high-dose Anemone tomentosa groups, miRNA-146a mimics and inhibitor groups. After drug intervention, miRNA-146a-5p mRNA in prostasome was detected by real-time polymerase chain reaction, and TLR-4, inhibitor of NF-κB (IκB), phosphorylation of IκB (p-IκB) and tumor necrosis factor receptor-associated factor 6 (TRAF6) protein were detected by western blot, and inflammatory cytokines were detected by enzyme-linked immunosorbent assay. Results Compared with normal group, the expression level of miRNA-146a-5p mRNA in prostasome was down-regulated in each of the other six groups (P