OBJECTIVE: This study aimed to investigate the effect of Sox3 expression on biological behaviors of esophageal squamous cell carcinoma (ESCC) and explore its possible mechanism. METHODS: ESCC cell lines that highly expressed Sox3 were selected and transfected with lentivirus carrying sox3 siRNA to establish ESCC cell lines which expressed Sox3 of different levels. Using in vitro experiments including cell invasion, cell scratch, cell proliferation and tube formation of lymphatic endothelial cells, as well as an in vivo experiment of axillary lymph node metastasis in a nude mouse model of a xenotransplanted tumor, the effect of Sox3 expression variation on lymphangiogenesis and lymph node metastasis in ESCC cells was investigated. In addition, ELISA, Western blot and immunohistochemical methods were used to study the regulatory effects of Sox3 on relevant molecules such as VEGF-C/D and to explore the potential mechanisms that affected lymphatic metastasis. RESULTS: The high expression of Sox3 in ESCC cells in vitro could significantly promote the proliferation, invasion, migration and tube formation of lymphatic endothelial cells. High expression of Sox3 in vivo could significantly promote lymph node metastasis of ESCC cells, and we have demonstrated that the upregulation of Sox-3 expression could promote the expression and secretion of VEGF-C and VEGF-D both in vivo and in vitro. After blocking the VEGFR-3 receptors on lymphatic endothelial cells, the effect of Sox3 on promoting lymphangiogenesis has decreased significantly, confirming that Sox3 acts through VEGF-C/D to promote lymphangiogenesis. CONCLUSIONS: It is suggested that Sox3 possibly induces lymphangiogenesis by increasing the expression of VEGF-C/D in ESCC cells, thereby promoting the lymph node metastasis of the tumor. Thus, Sox-3 may become a new prognostic marker and therapeutic target in ESCC.