Further, we demonstrated that miR-218 inhibited cell proliferation and arrested cell cycle at G1 phase by directly targeting 3ʹUTR of ROBO1. In summary, the results indicate that repression of miR-218 plays an essential role in ESCC tumorigenesis, which is at least partly due to CpG hypermethylation. This evidence concerns the gene ROBO1 and esophageal squamous cell carcinoma.