MicroRNAs (miRNAs) are important regulators and associated with the development of many different types of cancer, including gastric cancer. However, their pathophysiologic role and their relevance to tumorigenesis, invasion and metastasis are still unknown. In our current study, we performed microRNA array and found that 28 of miRNAs were differentially expressed in INF type of gastric cancer. Among 28 miRNAs, miR-29b was one of the most significantly down-regulated miRNA. Further bioinformatics analysis showed that MMP2 was a potential target of miR-29b. Interestingly, luciferase analysis showed that miR-29b negatively regulates MMP2 by binding with the miRNA response element (MRE) on the 3'UTR of MMP2. In addition, overexpression of miR-29b significantly decreased the mRNA and protein level of MMP2 and the activity of MMP2 to suppress gastric cancer cell migration. Moreover, lentivirus mediated overexpression of miR-29b dramatically suppressed the ability of BGC823 cells to form colonies and their ability to develop tumor in nude mice. Finally, our qPCR and western blot analysis showed that miR-29b was significantly reduced in clinical gastric cancer tissue, whereas MMP2 protein was significantly up-regulated, suggesting that this aberrant down-regulation of miR-29b might be associated with the abnormal regulation of MMP2 and the development of gastric cancer. Significant apparent was also found between miR-29b expression and TNM staging, lymph node status, tumor differentiation and Ming classification. Together, our data suggest an important regulatory role of miR-29b in the development of gastric cancer. Thus, miR-29b and MMP2 might be important diagnostic or therapeutic targets for human tumor diseases.