Background: Cancer stem cells (CSCs) accelerate the growth of hepatocellular carcinoma (HCC) residual after incomplete radiofrequency ablation (In-RFA). The present study aimed to detect the effects of In-RFA on stemness transcription factors (STFs) expression which are important for the production and function of CSCs, and to find which STFs promote HCC stemness after In-RFA. Methods: HepG2 cells were used for in vitro and in vivo studies. Flow cytometry and sphere-formation assays were used to detect the level and function of CD133(+)CSCs in the models. PCR array and ELISA were applied to analyze the altered expression of 84 STFs in CD133(+)CSCs in two models. Specific lentiviral shRNA was used to knockdown STFs expression, followed by detecting In-RFA's effects on the levels and function of CD133(+)CSCs. Results: In-RFA was identified to induce CD133(+)CSCs and increase their tumorigenesis ability in vitro and in vivo. The mRNA levels of 84 STFs in CD133(+)CSCs were detected by PCR array, showing that 15 and 22 STFs were up-regulated in two models, respectively. Meanwhile, the mRNA levels of seven common STFs were up-regulated in both models. ELISA assay demonstrated that only the protein of sex determining region Y-box 9 (SOX9) was up-regulated in both models, the protein levels of the other 6 common STFs did not increase in both models. Finally, SOX9 was identified to play an important role in inducing, maintaining stemness and promoting tumorigenesis ability of CD133(+)CSCs in both models. Conclusion: In-RFA-induced SOX9 stimulates CD133(+)CSCs proliferation and increases their tumorigenesis ability, suggesting that SOX9 may be a good target for HCC treatment. (C) 2018 First Affiliated Hospital, Zhejiang University School of Medicine in China. Published by Elsevier B.V. All rights reserved.