Accumulating evidences indicated that some microRNAs (miRNAs) play a critical role during the carcinogenesis. In the present study, we found that miR-124 is down-regulated in esophageal squamous cell carcinoma (ESCC) tissues. Three miR-124 encoding genes, including mir-124-1, mir-124-2, and mir-124-3, harboring CpG islands undergo methylation-mediated miR-124 inactivation in ESCC tissues. The methylation status of all these three genes was negatively associated with the expression of miR-124. The low expression of miR-124 and the hypermethylation of mir-124-1 and mir-124-3 were associated with the clinico-pathological parameters indicating the poor prognosis. In addition, promoter methylation of all three genes plus low expression of miR-124 was the independent poor prognostic marker for ESCC patients. In conclusion, miR-124 may function as a tumor suppressive miRNA, and hypermethylation-mediated inactivation of miR-124 may be useful for a poor prognostic marker for ESCC patients.