Purpose: The BTF3 is involved in oncogenesis, while the biological roles in HSCC remain unclear. The aim of this study was to explore the impact of BTF3 knockdown on biological phenotypes of human HSCC in vivo and in vitro. Methods: The expression of BTF3 was assessed in HSCC and normal tissues. In vitro experiments were performed to explore impact of BTF3 knockdown on biological phenotypes of human HSCC cell line, including proliferation, cell cycle, and apoptosis. Moreover, nude mice were used to evaluate growth of xenograft tumors. Finally, gene chip was used to explore the potential signaling pathways of BTF3, with confirmation of potential BTF3-related genes. Results: Our results showed elevated expression of BTF3 was observed in HSCC tumors compared to paired adjacent normal tissues in 68 patients, and positively associated with lymph node metastasis and survival of this HSCC patient cohort. In addition, in vitro experiments showed that BTF3 knockdown significantly impaired regulation of proliferation, cell cycle, and apoptosis, potentially via ATM signaling pathway. Finally, in vivo experiments demonstrated that BTF3 functioned as an oncogene by promoting the development and progression of HSCC tumors, indicating its oncogenic role in HSCC. Conclusions: This study for the first time demonstrated that expression of BTF3 is upregulated in HSCC tumors and this upregulation is positively correlated with lymph node metastasis of this malignancy. The oncogenic role of BTF3 is further validated for tumor promotion and progression of HSCC in vivo, indicating that BTF3 is a potential therapeutic target and prognostic biomarker for HSCC.