Silencing of protein-coding tumor suppressor genes (TSGs) by CpG island hypermethylation is a common occurrence in gastric cancer (GC). Here, we examine if tumor suppressor microRNAs (miRNAs) are silenced in a similar manner. Real-time quantitative PCR (RTQ-PCR) was employed to investigate the expression level of four candidate miRNAs in GC tissues (n=30) and cell lines. Basing on RTQ-PCR results and bioinformatics approach, miR-9 was chosen for further study on epigenetic regulation. Bisulfite genomic sequencing PCR (BSP) was performed to assess the methylation status of miR-9 in GC tissues. In both GC cell lines and animal models, demethylation was performed either by treatment with 5-aza-2'-deoxycytidine (5-AZA-CdR) or by siRNA targeting DNMT1. We also analyzed the relationship between miRNAs and several clinicopathological features. Candidate miRNAs (miR-9, miR-433, miR-19b, and miR-370) were found strongly downregulated in GC tissues and cell lines. Their expression was increased following 5-AZA-CdR treatment. CpG island methylation of miR-9 was significantly higher in GC tissues compared to normal controls. After two demethylation treatments, miR-9 methylation degree was significantly decreased and miR-9 expression was ob-viously restored in GC cells and animal models. Deregulation of miR-9 was positively correlated with tumor lesion size. Three other miRNAs, miR-19b, miR-433, and miR-370 were assοciated with lymph node metastasis, decreased curvature, and poorly differentiated carcinoma. miR-19b and miR-433 were positively correlated with male gender. Of four candidate miRNAs downregulated in GC, miR-9 is epigenetically regulated by DNA methylation both in vitro and in vivo.