Background: Esophageal squamous cell carcinoma (ESCC) is a highly lethal cancer, and its underlying molecular mechanisms are poorly understood. Recent large-scale genome-wide association studies in Chinese Han populations have identified an ESCC susceptibility locus within the SLC39A6 gene. Here, we sought to explore the expression and biological function of SLC39A6 in ESCC. Methods: Multiethnic validation of SLC39A6 protein expression was performed in different cohorts of patients from Chinese Han and Kazakh populations in the Xinjiang region by immunohistochemistry. The associations among SLC39A6 expression, clinicopathological parameters, and prognosis outcomes of ESCC were analyzed. And the effects of SLC39A6 silencing by siRNA on cell proliferation, apoptosis, and invasiveness, as well as the proteins involved in epithelial-to-mesenchymal transition (EMT) of esophageal cancer cells, were studied. Results: SLC39A6 protein expression increased progressively from normal esophageal epithelium (NEE) to low-grade intraepithelial neoplasia to ESCC, and finally reached the highest in high-grade intraepithelial neoplasia from Han ethnic. Similarly, SLC39A6 protein was significantly overexpressed in Kazakh ethnic ESCC compared with that in NEE. Increased expression of SLC39A6 was found to be closely correlated with histological grade and early Tumor-Node-Metastasis stage I/II. High tumorous SLC39A6 expression was significantly correlated with shorter overall survival (OS). Cox regression analysis confirmed that SLC39A6 expression was an independent prognostic factor for poor OS in ESCC. Experimentally, the suppression of SLC39A6 expression promoted ESCC cell apoptosis but abrogated proliferation and invasion, and induced an EMT phenotype that included enhanced expression of E-cadherin, loss of vimentin, and morphological changes in ESCC cells in vitro. Conclusions: Combined, our findings highlight a tumor-promoting role for SLC39A6 in ESCC, suggesting that SLC39A6 could serve as an early detector of high-risk subjects and prognostic biomarker. The targeting of SLC39A6 might be a potential therapeutic strategy for blocking ESCC.