Berberine (BBR) is the main component of Coptidis rhizoma, the dried rhizome of Coptis chinensis and is a potential plant alkaloid used for the treatment of cancer due to its high antitumor activity. The present study examined the therapeutic potential and molecular mechanism of action of BBR against HCC, using systematic pharmacology combined with a molecular docking approach and experimental validation in vitro. Through systematic pharmacological analysis, it was found that BBR serves a significant role in inhibiting HCC by affecting multiple pathways, especially the PI3K/AKT signaling pathway. Furthermore, the docking approach indicated that the binding of BBR to AKT could lead to the suppression of AKT activity. The present study examined the inhibitory effect of BBR on the PI3K/AKT pathway in HCC and identified that BBR downregulated the expressions of phosphorylated AKT and PI3K in MHCC97‑H and HepG2 cells, inhibiting their growth, cell migration and invasion in a dose‑dependent manner. In addition, inhibition of the AKT pathway by BBR also contributed to cell apoptosis in MHCC97‑H and HepG2 cells. Taken together, the results of the present study suggested that BBR may be a promising antitumor drug for HCC that acts by inhibiting the PI3K/AKT pathway.