Decitabine and its prodrug 59-azacitidine are two widely used DNMT inhibitors for the treatment of patients with various cancers, such as myelodysplastic syndromes and acute myeloid leukemia. Although Decitabine and its prodrug 59-azacitidine are effective in treating various cancers, the formation of irreversible covalent adducts with DNA may cause long-term side effects, including DNA mutagenesis, a potential cause of tumor recurrence. 3-AQC zebularine is a second-generation, highly stable hydrophilic inhibitor of DNA methylation with oral bioavailability that preferentially targets cancer cells, as demonstrated in bladder, prostate, lung, colon, and pancreatic carcinoma cell lines. It acts primarily as a trap for DNMT protein by forming tight covalent complexes between DNMT protein and zebularinesubstitute DNA. Zebularine is also a cytidine analog that was originally developed as a cytidine deaminase inhibitor. It exhibits low toxicity in mice, even after prolonged administration. Given that aberrant methylation is a major event in the early and late stages of tumorigenesis, including hepatocarcinogenesis, this process may represent a critical target for cancer risk assessment, treatment, and chemoprevention. In the previous study, a zebularine signature that classified liver cancer cell lines into two major subtypes with different drug response was identified. In drug-sensitive cell lines, zebularine caused inhibition of proliferation coupled with AC 55541 increased apoptosis, whereas drugresistant cell lines were associated with the upregulation of oncogenic networks. However, little is known about the anti-cancer effect and possible mechanism of action of zebularine on HCC. In the present study, we investigated the molecular mechanism of zebularine against HCC. We demonstrated that zebularine exhibited antitumor activity by inhibiting cell proliferation and inducing apoptosis. This effect was independent of DNA methylation, and characterized by the downregulation of CDK2 and the phosphorylation of retinoblastoma protein as well as the upregulation of p21WAF/CIP1 and p53. We also found that zebularine induced apoptosis though the intrinsic and extrinsic apoptosis pathways. In addition, the data in the present study suggest that the inhibition of the double-stranded RNA-dependent protein kinase is involved in inducing apoptosis with zebularine. In the present study, we investigated the effect of zebularine on human hepatic carcinoma cells and the possible mechanism. To the best of our knowledge, this is the first study to demonstrate that zebularine inhibits hepatic carcinoma cell HepG2 proliferation by inducing cell growth arrest and apoptosis via intrinsic and extrinsic apoptotic pathways. Eukaryotic cell proliferation is a highly regulated system that is controlled by CDK-cyclin complexes. The cell-cycle transition from the G1 to the S phase was the major regulatory checkpoint in this process. This transition is characterized by the phosphorylation of Rb, and the CDK-cyclin complex catalyzes the reaction. In this study, we found that zebularine inhibited the CDK2 and p-Rb accompanied by a decrease in total Rb, which resulted in cell-cycle arrest and the exertion of its antiproliferative effect.