The anti-prostate cancer activity and mechanism of different solvent extracts from Anemones Rivularis Radix (ARR) were studied by network pharmacology and experimental validation.ARR was extracted with water,75% ethanol,and ethyl acetate to obtain the crude extracts.The effects of three extracts on the proliferation and apoptosis of prostate cancer cells PC3 were analyzed by MTT and flow cytometry in vitro,respectively.Chemicals of ethyl acetate crude extracts were analyzed by GC-MS.The active constituents and anti-prostate cancer targets of ARR were analyzed by network pharmacology.PPI network was constructed,and core targets were screened by topological analysis.The anti-prostate cancer signaling pathway of extracts was analyzed by GO and KEGG enrichment analysis.Based on the above results,the mechanism of action was predicted.The expression of genes in extract-treated PC3 cells at the transcriptional level related to the TNF signaling pathway was assayed through RT-PCR,which further verified the predicted targets and mechanism.Results showed three extracts had strong inhibitory effect on PC3 cells proliferation,and induced apoptosis (P<0.01) significantly.Compounds betulinic acid and huzhangoside D were the main active ingredients of ARR against prostate cancer based on GC-MS and network pharmacology.A total of 89 targets related to anti-prostate cancer were obtained,and 15 core targets were selected further,including JUN,BCL2L1 and HSP90AA1.GO functional enrichment showed there were 142 biological processes,28 cell compositions,and 41 molecular functions involved.The intersection targets mainly involved TNF,TRP,NF-κB,and cancer pathway abased on KEGG analysis.Results of RT-PCR showed the expression of key genes in the TNF signaling pathway were affected by three extracts significantly,which was consistent with the results predicted by the network pharmacology.This study revealed three extracts of ARR had strong inhibitory activity against prostate cancer,and betulinic acid and huzhangoside D were the main active compounds.The mechanism of action may be related to the regulation of TNF,TRP,NF-κB and cancer pathways.