MicroRNAs (miRNAs) operate as tumor suppressor or carcinogen to regulate cell proliferation, metastasis, inva- sion, differentiation, apoptosis, and metabolic process. In the present research, we investigated the effect and mechanism of miR-496 in human gastric cancer cells. miR- 496 was downregulated in two gastric cancer cell lines, AGS and MKN45, compared with normal gastric epithelial cell line GES-1. miR-496 mimics inhibited the proliferation of AGS cells after the transfection for 48 and 72 h. The migra- tion and invasion of AGS cells were also inhibited by the transfection of miR-496 mimics. miR-496 mimics induced the apoptosis through upregulating the levels of Bax and Active Caspase 3 and downregulating the levels of Bcl-2 and Total Caspase 3. Bioinformatics analysis showed that there was a binding site between miR-496 and Lyn kinase (LYN). miR-496 mimics could inhibit the expression of LYN in AGS cells. LYN overexpression blocked the inhibition of tumor cell growth, as well as the inhibition of AKT/mTOR signaling pathway induced by miR-496. In conclusion, miR- 496 inhibited the proliferation through the AKT/mTOR sig- naling pathway via targeting LYN in gastric cancer cells. Our research provides a new potential target for clinical diagnosis and targeted treatment for gastric cancer.