Our study aimed to investigate the isoform-specific distribution of 14-3-3 in tongue squamous cell carcinoma (TSCC) and their association with cancer progression, and to further discuss their roles in cancer cell survival. In this study, 42 TSCC specimens and their matched normal para-carcinoma sections were collected. The immunohistochemistry analysis identified that 14-3-3σ and ζ \\isoforms presented significantly higher expression in cancerous tissues compared with the matched normal tongue tissue sections. 14-3-3ζ expression was associated with tumor T stage, lymph node metastasis and poor prognosis of TSCC. In vitro study revealed that 14-3-3ζ silencing alleviated the proliferation and migration of TSCC cells while promoted cancer cell apoptosis. 14-3-3ζ could bind to and inactivate FOXO3a transcription factor, in turn leading to the movement of the 14-3-3ζ-FOXO3a complex from nucleus to cytoplasm, which was inhibited after 14-3-3ζ silencing. Both 14-3-3ζ and FOXO3a silencing increased caspase 3 and 9 activation, while reduced inner mitochondrial membrane potential. Collectively, 14-3-3ζ may serve as a hallmark and prognostic marker of TSCC. 14-3-3ζ can bind to the FOXO3a transcription factor to promote the export of the complex to the cytoplasm, leading to enhanced proliferation and migration of tongue cancer cells.