Objective: MicroRNAs (miRNAs) play important roles in carcinogenesis. The aim of the present study was to explore the effects of miR-181b on gastric cancer. Methods: The expression level of miR-181b was quantified by qRT-PCR. MTT, flow cytometry and matrigel invasion assays were used to test proliferation, apoptosis and invasion of miR-181b stable transfected gastric cancer cells. Results: miR-181b was aberrantly overexpressed in gastric cancer cells and primary gastric cancer tissues. Further experiments demonstrated inducible expression of miR-181b by Helicobacter pylori treatment. Cell proliferation, migration and invasion in the gastric cancer cells were significantly increased after miR-181b transfection and apoptotic cells were also increased. Furthermore, overexpression of miR-181b downregulated the protein level of tissue inhibitor of metalloproteinase 3 (TIMP3). Conclusion: The upregulation of miR-181b may play an important role in the progress of gastric cancer and miR-181b maybe a potential molecular target for anticancer therapeutics of gastric cancer.