Abstract: Objective To investigate the biological functions of miR-100 in esophageal squamous cell carcinoma(ESCC) cell. Methods miR-100 was cloned into a GFP expressing vector, which was further transfected into esophageal squamous cell carcinoma cell line Ec-109 by Lilpofectamine, and GFP positive cells were then selected by flow cytometry. Cell cycle, apoptosis and migration were analyzed by flow cytometry, wound healing and Transwell assay, respectively, upon the expression of miR-100 in Ec-109 cells. Results The high expression of miR-100 could induce G₁ arrest: the percentage of cells number were increased in the G₁ phase, while cells in the S or G2/M were decreased. Under the serum free culture condition, the presence of miR-100 could enhance cell apoptosis and also suppress Ec-109 cells migration. Conclusion The exogenous expression of miR-100 exhibits a tumor suppressor role in esophageal squamous cell carcinoma cell line Ec-109, including inducing G₁ arrest, suppressing cell migration and promoting cell apoptosis.