Esophageal cancer (EC) is a common cancer worldwide. Sine oculis homeobox homolog (SIX3) is a human transcription factor that regulates the progression of vertebrate eye and fetal forebrain. However, studies on the function of SIX3 in human tumorigenesis remain rare. In this study, we aim to evaluate the role and the significance of SIX3 in EC. The TCGA database and clinical samples were used to assess the expression of SIX3 in EC patients. The Kaplan-Meier method and Cox's proportional hazards model were performed to analyze the correlations between SIX3 expression and EC clinical outcomes. The expressions of SIX3 in EC cells were measured by quantitative reverse transcription PCR analysis. The cell proliferation was detected using cell counting kit-8 and colony formation assay. The migration and invasion capacity of EC cells were evaluated using wound healing and Transwell methods. Western blot assay was used to measure the alterations in some important protein expression levels in the PI3K/Akt signaling pathway. We found that SIX3 was highly expressed in the EC tissues and cells. In addition, high expression of SIX3 was related to poor survival. The knockdown of SIX3 significantly inhibited the proliferation, migration, and invasion of ECA109 cells. A similar pattern was also found in the proliferation and migration of SKGT-4 cell line. The expression levels of some key proteins in the PI3K/Akt signaling pathway were obviously decreased after cells were transfected with si-SIX3, possibly resulting in PI3K/AKT signaling inactivation. In addition, E-cadherin and N-cadherin showed some change. Collectively, the results shed light on a potentially promoting role of SIX3 in human EC. Thus, SIX3 might be considered a novel prognostic biomarker and therapeutic target for EC patients.