Sam68 (Src-associated in mitosis of 68 kD) is a KH domain RNA-binding protein. The expression of Sam68 was correlated with kinds of tumors. Yet, the expression mechanisms and physiological significance of Sam68 in ESCC remains unclear. In this study, we clarified a potential role of Sam68 in the treatment of ESCC. Western blot and immunohistochemistry (IHC) analysis revealed that the protein level of Sam68 was higher in ESCC tumor tissues and cell lines. In addition, IHC stain revealed that Sam68 was positively correlated with clinical pathologic variables such as tumor grade and tumor invasion. In addition, Sam68 could be an independent prognostic indicator for patients' overall survival. In vitro studies such as starvation and refeeding assay along with Sam68-shRNA transfection assay demonstrated that Sam68 expression promoted proliferation of ESCC cells. And Sam68 downregulation caused decreased rate of cell growth and colony formation. Reasons are associated with growth arrest of cell cycle at G1/S phase. Moreover, our results clarified that Sam68 could promote ESCC cell proliferation via the activation of Akt/GSK-3β pathway. This research indicated that Sam68 might accelerate the cell cycle progression and be considered as a new therapy target in ESCC.