5-Azacytidine (5-azaC) was originally identified as an anticancer drug (NSC102876) which can causehypomethylation of tumor suppressor genes. To assess its effects on runt-related transcription factor 3 (RUNX3),expression levels and the promoter methylation status of the RUNX3 gene were assessed. We also investigatedalteration of biologic behavior of esophageal carcinoma TE-1 cells. MTT assays showed 5-azaC inhibited theproliferation of TE-1 cells in a time and dose-dependent way. Although other genes could be demethylated after5-azaC intervention, we focused on RUNX3 gene in this study. The expression level of RUNX3 mRNA increasedsignificantly in TE-1 cells after treatment with 5-azaC at hypotoxic levels. RT-PCR showed 5-azaC at 50 μMhad the highest RUNX3-induction activity. Methylation-speciﬁc PCR indicated that 5-azaC induced RUNX3expression through demethylation. Migration and invasion of TE-1 cells were inhibited by 5-azaC, along withgrowth of Eca109 xenografts in nude mice. In conclusion, we demonstrate that the RUNX3 gene can be reactivatedby the demethylation reagent 5-azaC, which inhibits the proliferation, migration and invasion of esophagealcarcinoma TE-1 cells.