miR-516b, as a tumor suppressor in several tumors, its regulatory role in esophageal squamous cell carcinoma (ESCC) hasn't been previously reported.This study was to investigate the potential role of miR-516b in ESCC.miR-516b expression was measured in ESCC tumor specimens and matched adjacent non-cancerous tissues from 80 ESCC patients. The association between miR-516b and clinicopathological features of these patients was analyzed. The effect of miR-516b was evaluated by cell proliferation, migration, invasion and apoptosis assays in ESCC cell line EC9706 and TE-9. The role of miR-516b in vivo was further studied by constructing ESCC xenograft mice model. The direct target of miR-516b was predicted by public miRNA database and confirmed by luciferase reporter assay. The regulation of miR-516b on the target gene was further confirmed in vitro and in vivo. The expressions of proteins related to cell cycle and apoptosis were analyzed by western blot analysis, and cell migration and invasion were assessed by transwell assays.miR-516b expression was reduced in ESCC tissues and cells, and correlated with advanced TNM stage, depth of invasion, lymphatic metastasis and poorer overall survival in ESCC patients. miR-516b was upregulated by miR-516b mimics repressing cell proliferation, and inducing G1 cell cycle arrest and apoptosis. miR-516b upregulation also suppressed the growth of ESCC xenograft tumor in nude mice and the invasion of ESCC cells via regulating the epithelial-mesenchymal transition pathway. CCNG1 was identified as a direct downstream target of miR-516b.The results demonstrated miR-516b functions as a tumor suppressor by directly modulating CCNG1 expression in ESCC cells, and may be a novel therapeutic and prognostic biomarker for ESCC.