Japonicoside B (JaB), a representative constituent of the traditional Chinese medicinal plant Smilacina japonica A. Gray, was investigated for its effects on human hepatocellular carcinoma SMMC-7721 and HepG2 cells. This study was designed to explore the anti-hepatocellular carcinoma effects and underlying mechanisms of JaB by observing its impact on SMMC-7721 and HepG2 cells, as well as its regulatory role in the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway. Multiple experimental approaches were employed, MTT assay to assess proliferation inhibition effect, gene chip screening for pathway identification, fluorescence staining for morphological observation, flow cytometry for apoptosis and cell cycle analysis, complemented by Western blot and RT-PCR to evaluate protein and mRNA expressions. Results demonstrated that JaB exhibited potent anti-proliferative activity against both SMMC-7721 and HepG2 cells. The gene chip screening revealed that the anti-hepatocellular carcinoma activity of JaB was mainly manifested as the inhibition of cell proliferation, which was associated with the PI3K/AKT signaling pathway. Further investigations through fluorescence staining, flow cytometry, Western blot, and RT-PCR confirmed that JaB exerted anticancer effects by modulating PI3K/AKT signaling, leading to proliferation inhibition, apoptosis induction, and cell cycle arrest. Collectively, this study reveals that JaB exerts anti-hepatocellular carcinoma effects through PI3K/AKT pathway regulation, effectively inducing apoptosis and cell cycle arrest in SMMC-7721 and HepG2 cells.