Chibby (or PIGEA-14) is a novel antagonist of the Beta-catenin pathway in nucleus. However, the tumor-suppressing function of Chibby and the importance of nuclear targeting to the cellular functions of Chibby have not been validated. By fusion of Chibby cDNA with green fluorescent protein (GFP) or Flag-tag, it was found that exogenous Chibby expression was detected in the nucleus as well as cytoplasm of transfected HeLa cells, but with a preferential nuclear localization (more than 50% cells with nuclear Chibby expression). Chibby overexpression significantly abrogated the cellular Beta–catenin activities and induced apoptosis in HeLa cells. Moreover, Chibby gene delivery attenuated the proliferation, migration, and anchorage-independent growth of HeLa cells, supporting the tumor suppressor function of Chibby. Mutation or deletion of the predicted nuclear localization sequence (NLS), at residues 123-126, significantly promoted the cytoplasmic localization of Chibby, indicating residues 123-126 is the NLS domain of Chibby. Interestingly, ecotopic expression of Chibby NLS mutants remained capable of inducing apoptosis and inhibiting Beta–catenin activities in HeLa cells. Besides, overexpression Chibby NLS mutants effectively attenuated the viability, motility and colonies formation of HeLa cells. Expression analysis revealed that Chibby NLS mutants retained Beta-catenin in the cytoplasm and prevented its nuclear entry, thereby inhibiting the Beta-catenin transcriptional activities. In summary, Chibby shuttles between nucleus and cytoplasm, and possesses the functions of tumor suppressor and Beta-catenin antagonist.