Runt-related transcription factor 3 (RUNX3) is a tumor suppressor gene whose reduced expression may playan important role in the development and progression of esophageal squamous cell cancer (ESCC). The aim ofthis study was to investigate the clinical relevance of RUNX3 in ESCC patients and effects of overexpressionon biological behaviour of Eca109 cells in vitro and in vivo. Immunohistochemistry was performed to detect theclinical relevance of RUNX3 and lymph node metastasis in 80 ESCC tissues and 40 non-cancerous tissues usingthe SP method. RT-PCR and Western blotting were applied to assess the RUNX3 level and verify the Eca109 cellline with stable overexpression. Localization of RUNX3 proteins was performed by cell immunofluorescence.CCK-8 and Scrape motility assays were used to determine proliferation and migration and the TUNEL assayto analyze cell apoptosis. Invasive potential was assessed in cell transwell invasion experiments. In nude mice,tumorigenesis in vivo was determined. Results showed decreased expression of RUNX3 in esophageal tissue tobe significantly related to lymph node metastasis (LNM) (P<0.01). In addition, construction of a recombinantlentiviral vector and transfection into the human ESCC cell line Eca109 demonstrated that overexpressioncould inhibit cell proliferation, migration and invasion, and induce apoptosis. The in vivo experiments in miceshowed tumorigenicity and invasiveness to be significantly reduced. Taken together, our studies indicate thatunderexpression of RUNX3 in human ESCC tissue is significantly correlated with progression. Restoration ofRUNX3 expression significantly inhibits ESCC cells proliferation, migration, invasion and tumorigenesis.