Human epidermal growth factor receptor 2 (HER2) overexpression is not only closely associated with the tumor growth, but is also related to tumor invasion. We here aimed to investigate the mechanism of HER2 mediation in the pathogenesis of gastric cancer. The human gastric cancer cell lines SGC-7901, MKN-45, AGS, the immortalized cell line GES-1 derived from normal gastric mucosa. Cell transfection and selection of stable cell lines and the gene and protein levels of HER2 and Matrix metalloproteinase-9 (MMP-9) were examined to determine the molecular relationship between them in the pathogenesis of gastric cancer. The human gastric cancer cell lines SGC-7901, MKN-45, AGS, the immortalized cell line GES-1 derived from normal gastric mucosa. Cell transfection and selection of stable cell lines and the gene and protein levels of HER2 and MMP-9 were examined to determine the molecular relationship between them in the pathogenesis of gastric cancer. We demonstrated that vector-based shRNA significantly knocked down the expression of HER2 and considerably inhibited both the migration and invasion of gastric cancer cells. HER2 knockdown resulted in the downregulation of the expression of MMP-9, whereas HER2 overexpression improved the transcription of MMP-9 through the activation of an MMP-9 promoter. The promoter region of MMP-9 between -2500 and -2000 bp was found to be crucial for the upregulation of HER2-mediated transcription. Furthermore, a truncated promoter (-70 to +63) did not display any transcriptional activity. Cell invasion activity was almost completely inhibited when MMP-9 was knocked down. Conversely, the overexpression of MMP-9 partly rescued the invasion ability of cell strains with knockdown HER2. These findings help further understanding of the molecular mechanisms through which HER2 promotes malignancy, and suggest that targeting both HER2 and MMP-9 may be required to effectively block HER2 signaling in gastric cancer therapy.