Gastric cancer (GC) is a complex heterogeneous process and the molecular mechanisms underlying its initiation or propagation are still not very well characterized. Aberrant gene expression are key features of cancer. DNA methylation in a promoter region is an important epigenetic mechanism for the gene silencing. Here, the impact of DNA methylation in regulating the expression of miR-125b1 is explored. A total of 285 genetically unrelated subjects including 175 healthy controls and a total of 110 GC patients participated in this study. we performed nested methylation-specific polymerase chain reaction (MS-PCR) to evaluate methylation pattern of miR-125b1 promoter and quantitative real-time polymerase chain reaction (qRT-PCR) to determine the RNA expression changes in GC and normal tissues. The frequency of methylated allele was 24.5% in GC cases but only 10% in normal tissues. Statistically significant correlation between CpG dinucleotide methylation of miR-125b1 promoter and increased risk of gastric adenocarcinoma was observed (OR=2.57; 95%CI 1.60-4.13; P=0.0001). In addition, miR-125b1 promoter methylation correlated with tumor location and stages. miR-125b1 expression was much higher in normal tissue compared to cancerous tissue. However, methylation status of the miR-125b1 promoter was not correlated with miR-125b1 expression in cancerous specimens (p<0.05). In conclusion, this is a first report of miR-125b1 promoter methylation in GC. More research is needed to fully elucidate the underlying molecular mechanisms of GC susceptibility.