Gastric cancer is a most common malignancy and the third leading cause of cancer mortality worldwide. So it is important to identify the prognostic markers and effective therapeutic targets against gastric cancer. miRNA plays an important role in tumor cell cycle, differentiation, apoptosis, invasion and metastasis. Many studies devote to the mechanism of miRNA regulates gastric cancer carcinoma and progression. In the present study, we found that the level of miR-518 in gastric cancer and cell lines were lower than the control or the adjacent tissues by qRT-PCR. Transfected with miR-518 mimic trigger apoptosis in MKN45 and HGC27 gastric cancer cell in vitro and in vivo. Moreover, we found that MDM2 was negatively regulated by miR-518 via targeting 26–32 site of 3’UTR using luciferase reporter assay. The western blot assay showed that miR-518 up-regulated the expression of p53, pro-apoptotic protein Bax and active the activity of cleaved caspase-3, down-regulated expression of anti-apoptotic protein Bcl-2 via targeting MDM2. Thus, our study suggested that miR-518 acted as a new tumor suppressor by targeting MDM2 gene and trigger apoptosis in vivo and in vitro. The findings of the study first established the role of miR-518 in gastric cancer and may be a potential therapeutic target against gastric cancer in the further.