Background: This investigation was planned to evaluate the anti-cancer effects of royal jelly (RJ) obtained from Apis mellifera compared with doxorubicin as an anthracycline with potent anti-cancer activity and its cellular mechanisms against the human hepatoma cell line HepG2. Materials and Methods: The cytotoxic effects of various concentrations of RJ on the HepG2 cell viability by the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay were studied. For the primary and late apoptosis in HepG2 cells exposed with RJ, we used the Annexin-V (AV) assay using cytometry analysis using the commercial kit as explained by the manufacturer's guidelines. Real-time polymerase chain reaction assessed the gene expression of miRNA-34a (miR-34a), caspase-3, Bcl-2, and Bax. We also used the western blot to evaluate the protein expression levels of poly (ADP-ribose) polymerases (PARP), Caspase-3, Caspase-9, Bcl-2, and Bax. Results: The IC50 value of RJ was found 1.13 mg/mL for HepG2 cells. RJ revealed no cytotoxicity on normal THLE-3 cells with IC50 >2 mg/ml. RJ at the concentrations of ½ IC50 significantly increased (p < 0.05) apoptotic and necrotic cells from 0.96% to 28.3% and 9.3%, respectively. RJ at the concentration of IC50 significantly increased (p < 0.05) apoptotic and necrotic cells from 0.96% to 39.2% and 14.12%, respectively. The expression of miR-34a, Caspase-3, and the Bax gene was considerably (p < 0.001) up-regulated as they are dose-dependent, whereas the expression level Bcl-2 was considerably (p < 0.05) declined in the HepG2 cells exposed with RJ. Treatment of HepG2 cells treated with RJ triggered a significant inhibition of Bcl-2 protein, whereas a significant rise in PARP, Caspase-3, Caspase-9, and Bax expression was observed. Conclusion: Our results showed the promising anti-cancer effects of RJ against HepG2 cells, whereas the induction of apoptosis by various pathways is considered the main mechanism underlying the cytotoxic effect of RJ against HepG2 cells. The present study's findings propose that RJ can be a candidate agent for treating human HCC.