Emerging evidence suggests that Nanog is involved in cervical tumorigenesis. However, the regulating role of Nanog in tumorigenesis and chemosensitivity are still poorly understood. In this study, Nanog was disrupted by transcription activator-like effector nucleases (TALEN) in Hela cells and its expression was significantly decreased in a single-cell derived sub-clone with biallelic mutations. The disruption of Nanog not only induced down regulation of some other core transcription factor genes for cell self-renewal, such as Oct4, Sox2 and FoxD3, but also led to the down regulation of some mesenchymal representative genes, vimentin and N-adherin, and up regulation of the epithelial gene, E-cadherin. In addition, the invasiveness and clonogenicity of the Hela cells were obviously affected, and surprisingly their sensitivities to anti-cancer drugs were also significantly increased in vitro. After Xenograft into nude mice, the growth volumes of the neoplasms from the Nanog disrupted Hela cells were significantly smaller compared with those from wild type ones. In conclusion, these results suggest that disruption of Nanog may reverse the status of epithelial-mesenchymal transition, which is critical in tumorigenesis, and alleviate chemoresistance, as well as their invasiveness, in cervical cancer cells.