Background: Hepatocellular carcinoma (HCC) is the most common form of liver cancer that occurs in hepatocytes. Although many chemical drugs, e.g., cisplatin, methotrexate, taxis, and doxorubicin are used to treat HCC, there have been numerous reports related to the side effects of these drugs (e.g., emerging drug resistance, bone marrow failure, and gastrointestinal disorders). These issues led scientists to search for the novel anti-cancer drugs, mainly in natural products with greater efficiency and less toxicity. The current survey was intended to assess the anti-cancer effects of queen bee acid (10-Hydroxy-2-Decenoic Acid, 10-HDA) and its cellular mechanisms against the human hepatoma cell line HepG2. Materials and Methods: The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay was used to evaluate the effect of 10-HDA on the viability of HepG2 cells. The initial and late apoptosis in the HepG2 cells treated with 10-HDA were assessed by the Annexin-V (AV) assay. The level of the gene and protein expression of some apoptosis genes (e.g., caspase-3, Bcl-2-associated X protein (BAX), and B-cell lymphoma protein 2 (Bcl-2)), Poly (ADP-ribose) polymerases (PARP), and miRNA-34a (miR-34a), were measured by real-time PCR and Western blot. Results: The obtained findings revealed that HepG2 cell viability was markedly reduced (p < 0.01) following exposure to 10-HDA in a dose-dependent matter. The calculated half maximal cytotoxic concentration (CC50) value of 10-HDA was 59.6 µg/mL for HepG2 cells, while this value for normal THLE-3 cells was 106.4 µg/mL. We found that 10-HDA markedly elevated (p < 0.01) the percentage of necrotic and apoptotic cells from 0.94 to 9.7 and 27.6%, respectively. The real-time PCR results showed that the expression levels of the caspase-3, Bax, and miR-34a genes were significantly (p < 0.001) elevated. Contrary to these results, a significant (p < 0.01) reduction in the expression level of the Bcl2 gene was observed. The levels of protein expression of Caspase-3, PARP, and Bax were markedly elevated following exposure of HepG2 cells to 10-HDA at ¼ CC50, ½ CC50, and CC50. The level of protein expression of Bcl-2 was markedly reduced following exposure of HepG2 cells to 10-HDA at ¼ CC50, ½ CC50, and CC50 (p < 0.01). Conclusion: The current results confirmed the potent in vitro cytotoxic effects of 10-HDA on HepG2 cells with no significant cytotoxic effects on normal cells. Although its mechanisms of action have not been fully studied, the induction of apoptosis via different pathways was determined as one of the principle mechanisms of action of 10-HDA against HepG2 cells. Nevertheless, additional surveys must be performed to clearly understand the mechanisms of action and safety of this fatty acid.