Objective To investigate the effect and mechanism by which agrimonia pilosa inhibits hepatocellular carcinoma (HCC). Methods Potential core targets of agrimonia pilosa active components against HCC were screened using TCMSP, UniProt, GeneCards, OMIM, TTD, and STRING databases. Core targets with elevated mRNA and protein expression levels in HCC tissue versus normal liver tissue were identified via the UALCAN database. Their correlation with overall survival (OS) in HCC patients was assessed using the Kaplan-Meier Plotter database to determine high-expression targets associated with poor prognosis. Differential protein expression of the selected targets between normal and HCC tissue was validated using the HPA database. Meanwhile, their expression levels across different clinical stages of HCC and their associations with progression-free survival (PFS), recurrence-free survival (RFS), and disease-specific survival (DSS) were further verified with UALCAN and Kaplan-Meier Plotter. Molecular docking between agrimonia pilosa active components and target proteins was performed using software such as AutoDock, and the result  were visualized by PyMOL. Cell experiments in vitro were conducted to evaluate the effects of agrimonia pilosa treatment on proliferation, apoptosis, cell cycle distribution, and the mRNA and protein expression levels of MAPK3, CDK2, CDK4, and secreted phosphoprotein 1（SPP1）in HepG2 cells were detecting by Western blotting and Real-time PCR.Results Bioinformatics analysis identified MAPK3, CDK2, CDK4, and SPP1 as core targets. The mRNA and protein expression of these targets was significantly higher in HCC tissue than those in normal tissue (P<0.05), and their high expression was significantly associated with poor prognosis (HR>1, P<0.05). Stage and survival analyses further confirmed that these targets were persistently overexpressed across the clinical stages of HCC. High expression of CDK2, CDK4, and SPP1 was significantly negatively correlated with PFS, RFS, and DSS (P<0.05), while high MAPK3 expression was significantly correlated with shortened DSS (P<0.05). Molecular docking analysis indicated that the active components of agrimonia pilosa possessed strong binding potential to MAPK3, CDK2, CDK4, and SPP1. Cell experiments showed that agrimonia pilosa treatment induced G0/G1 and G2/M phase arrest, decreased the colony formation rate, and increased the apoptosis rate in HepG2 cells (P<0.05). The mRNA expression of MAPK3, CDK2, CDK4, and SPP1 decreased. The protein expression level of extracellular signal-regulated kinase 1（ERK1）, phosphorylated ERK1（p-ERK1）, CDK2, SPP1, and CDK4 (in the low-dose group) was downregulated, and phosphorylated CDK2（p-CDK2）protein in the high-dose group also decreased (P<0.05). Conclusion Agrimonia pilosa inhibits HCC cell proliferation and induces apoptosis by downregulating the expression of MAPK3, CDK2, CDK4, and SPP1.