The therapeutic targets of berberine for hepatocellular carcinoma (HCC) and its detailed mechanisms remain unexplored. Here, an integration of network pharmacology, proteomic, bioinformatic and in vitro biochemical approach was proposed to reveal therapeutic targets and pathways underlying the antiproliferative activity of berberine against HepG2 cells. Results indicated that berberine caused the cytotoxicity and inhibited the growth of HepG2 cells with IC50 values ranging from 92 μM to 118 μM. Network pharmacology analysis revealed that targeting apoptosis and cell cycle pathways by berberine contributed to its antitumor efficacy against HCC. Proteomic analysis demonstrated that mitochondria-related apoptosis pathways were involved in the cytotoxic action of berberine, as evidenced by the expression of mitochondrial dysfunction-mediated proteins. Moreover, a total of 160 significantly altered proteins were screened, among which AKAP12 presented significantly increased levels under berberine treatment. Bioinformatic analysis of various public datasets showed that expression of AKAP12 in HCC liver tissues was downregulated, emphasizing its role as a tumor suppressor. Immunoblotting validated the increased levels of AKAP12, while co-immunoprecipitation identified its interaction with Cyclin D1. These data, together with flow cytometry analysis, suggested that AKAP12 mediated cell cycle arrest, thereby suppressing cell proliferation. Altogether, the antiproliferative action of berberine in HepG2 cells involves both apoptosis and cell cycle arrest. Regulating AKAP12 signalling by berberine might provide a promising strategy for HCC treatment. This journal is