Hepatocellular carcinoma (HCC) is highly resistant to traditional chemotherapeutic approaches, which causes difficulty in the development of effective drugs for the treatment of HCC. Berberine, a major ingredient of Rhizoma coptidis, is a natural alkaloid used in traditional Chinese medicine. Berberine exhibits potent antitumor activity against HCC due to its high efficiency and low toxicity. In the present study, we found that berberine sensitized HepG cells to NF-κB-mediated apoptosis. Berberine exhibited a significant antiproliferation effect on the HepG2 cells and promoted apoptosis. Both qRT-PCR and immunofluorescence staining revealed that berberine reduced the NF-κB p65 levels in HepG2 cells. Moreover, p65 overexpression rescued berberine-induced cell proliferation and prevented HepG2 cells from undergoing apoptosis. These results suggest that berberine inhibits the growth of HepG2 cells by promoting apoptosis through the NF-κB p65 pathway.