Abstract: Objective To study the effect of downregulation expression of Nanog on malignant behavior of cervical cancer HeLa cells. Methods Gene editing tool TALENs was employed to induce downregulation expression of Nanog, and Nanog mutation was evaluated by sequencing. RT-PCR and Western blot was used to detect the mRNA and protein expression level, respectively. Colony-formation assay, Transwell invasion assay, and chemotherapy sensibility assay was carried out to assess the capacity of colony-formation, invasion, and chemoresistance, respectively. Results TALENs successfully induced Nanog mutation and downregulated Nanog expression. Nanog mRNA and protein expression of Nanog-mutated monoclonal HeLa cells downregulated 3 times compared to thoses of wild-type HeLa cells (P<0.05). Additionally, significant weakened abilities of colony-formation, invasion, and chemoresistance in monoclonal HeLa cells were observed when compared to those of wild-type HeLa cells (P<0.05). Conclusion Nanog mutation attenuates the malignant behavior of HeLa cells. Importantly, downregulation or silencing of Nanog is promising to be a novel strategy for the treatment of cervical carcinoma.