Background: MicroRNA-381 (miR-381) has been reported to play suppressive or promoting roles in different malignancies. However, the expression level, biological function, and underlying mechanisms of miR-381 in gastric cancer remain poorly understood. Our previous study indicated that transmembrane protein 16A (TMEM16A) contributed to migration and invasion of gastric cancer and predicted poor prognosis. In this study, we found that miR-381 inhibited the metastasis of gastric cancer through targeting TMEM16A expression. Methods: MiR-381 expression was analyzed using bioinformatic software on open microarray datasets from the Gene Expression Omnibus (GEO) and confirmed by quantitative RT-PCR (qRT-PCR) in human gastric cancer tissues and cell lines. Cell proliferation was investigated using MTT and cell count assays, and cell migration and invasion abilities were evaluated by transwell assay. Xenograft nude mouse models were used to observe tumor growth and pulmonary metastasis. Luciferase reporter assay, western blot, enzyme linked immunosorbent assay (ELISA) and immunohistochemistry were employed to explore the mechanisms of the effect of miR-381 on gastric cancer cells. Results: MiR-381 was significantly down-regulated in gastric cancer tissues and cell lines. Low expression of miR-381 was negatively related to lymph node metastasis, advanced tumor stage and poor prognosis. MiR-381 decreased gastric cancer cell proliferation, migration and invasion in vitro and in vivo. TMEM16A was identified as a direct target of miR-381 and the expression of miR-381 was inversely correlated with TMEM16A expression in gastric cancer tissues. Combination analysis of miR-381 and TMEM16A revealed the improved prognostic accuracy for gastric cancer patients. Moreover, miR-381 inhibited TGF-beta signaling pathway and down-regulated epithelial-mesenchymal transition (EMT) phenotype partially by mediating TMEM16A. Conclusions: MiR-381 may function as a tumor suppressor by directly targeting TMEM16A and regulating TGF-beta pathway and EMT process in the development of progression of gastric cancer. MiR-381/TMEM16A may be a novel therapeutic candidate target in gastric cancer treatment.