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Paederus, belonging to the Coleoptera and Staphylinidae, can prevent cell mitosis and inhibit tumors. However, few studies exist on the mechanism of Paederus on inhibiting the growth of tumors. Consequently, the present study used network pharmacology, molecular docking and in vitro experiments explore the anti-Hepatocellular Carcinoma (HCC) mechanism of Paederus. Methods: Network pharmacology was used to predict the potential mechanisms through which Paederus acts against HCC. Therefore, the active compounds of Paederus were retrieved from the CNKI, Wanfang, PubMed and Web of Science databases. The targets for the active compounds were obtained from the Targetnet database, then the common targets and signaling pathways were obtained by Protein-protein interaction (PPI) network analysis, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. Thereafter, the compound-target and target-pathway networks were constructed. Finally, in vitro experiments using the SMMC-7721 HCC cells were used to further verify the predicted anti-HCC mechanism of Paederus. Results: The study screened 40 active compounds from Paederus, and 26 targets related to the anti-HCC mechanism of Paederus were predicted through network pharmacology. These targets were involved in the Nod-like receptor, MAPK, PI3K- Akt, TNF and AMPK signaling pathways. In addition, cell experiments showed that Paederus reduce the proliferation rate of SMMC-7721 HCC cells. Moreover, Paederus could down-regulated the expressions of the HSP90AA1, IRAK-4 and CTSB proteins in the Nod-like receptor signaling pathways in SMMC-7721 HCC cells. Conclusion: The study revealed the potential mechanism through which Paederus acts against HCC by combining network pharmacology, molecular docking, and experimental verification.