Chemoresistance presents a major obstacle to the efficacy of chemotherapeutic treatment of cancers. Using chemotherapeutic drugs to select drug-resistant cancer cells in hepatocellular carcinoma (HCC) and several other cancer cell lines, we demonstrate that chemoresistant cells displayed cancer stem cell features, such as increased self-renewal ability, cell motility, multiple drug resistance, and tumorigenicity. Octamer 4 (Oct4) messenger RNA (mRNA) levels were dramatically increased in chemoresistant cancer cells due to DNA demethylation regulation of Oct4. By functional study, Oct4 overexpression enhanced whereas Oct4 knockdown reduced liver cancer cell resistance to chemotherapeutic drugs in vitro and in xenograft tumors. It is known that the Oct4-TCL1-AKT pathway acts on embryonic stem cells and cancer stem cells in cell proliferation through inhibition of apoptosis. We further demonstrate that Oct4 overexpression induced activation of TCL1, AKT, and ABCG2 to mediate chemoresistance, which can be overcome by addition of the PI3K/AKT inhibitor; therefore, a direct pathway of Oct4-TCL1-AKT-ABCG2 or a combination of Oct4-TCL1-AKT with the AKT-ABCG2 pathway could be a potential new mechanism involved in liver cancer cell chemoresistance. Moreover, the clinical significance of the Oct4-AKT-ABCG2 pathway can be demonstrated in HCC patients, with a strong correlation of expression patterns in human HCC tumors. The role of the Oct4-AKT-ABCG2 axis in cancer cell chemoresistant machinery suggests that AKT pathway inhibition (PI3K inhibitors) not only inhibits cancer cell proliferation, but may also enhance chemosensitivity by target potential chemoresistant cells. Conclusion: Oct4, a transcriptional factor of pluripotent cells, can mediate chemoresistance through a potential Oct4-AKT-ABCG2 pathway.