To investigate the association of single-nucleotide polymorphism (SNP) in DNA methyltransferase 3B (DNMT3B) gene and the risk of gastric cancer (GC), we detected -149C>T and -579G>T in the promoter region of the DNMT3B gene by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing analysis. The DNMT3B genotype was determined in 259 gastric cancer patients and 262 healthy controls that were frequency matched for age and gender. Results showed that individuals with at least one -579G allele were also at significantly decreased risk of gastric cancer [odds ratio (OR), 0.43; 95% confidence interval (CI) 0.26-0.72] compared with those having a -579TT genotype. The -149C>T genotype distribution was irrelevant to the risk of gastric cancer (OR, 1.49; 95% CI, 0.17-17.94) in the studied Chinese population. In addition, data suggested that DNMT3B genetic polymorphism varied among different races, ethnic groups, and geographic areas.