Folate deficiency is associated with increased risk of many human diseases including several cancers. Folate receptor-alpha (FR-alpha) plays an important role in mediating influx of folates into cells and its expression can be induced by some environmental risk factors. Our previous study showed that decreased expression of FR-alpha occurs in human gastric cancer cell lines. In order to reveal the molecular mechanism of the difference of FR-alpha expression among these cell lines and analyze the relation between the mutations or/and polymorphisms of FR-alpha gene and gastric cancer, we screened the mutations of global FR-alpha gene in 12 gastric cancer cell lines and 28 gastric tumors and matched normal tissues by using PCR-DHPLC and DNA sequencing techniques, then further checked the exon2 of FR-alpha gene in 138 gastric tumors and matched normal tissues. Furthermore, we examined the relationship between two polymorphisms of FR-alpha A1314G and C1816delC and risk for gastric cancer in 296 cases and 354 age and sex-matched controls in northern China. We found that the allele frequency of FR-alpha1314A among cases was significantly higher than that among controls (0.186 vs. 0.143, P=0.014). Subjects with the A/G and A/A genotype had an increased risk for developing gastric cancer compared with the G/G genotype (OR=1.55; 95% CI=1.10-2.17). These results support the hypothesis that genetic polymorphism in the FR-alpha gene may contribute to susceptibility to carcinogenesis of the gastric cancer in the at-risk Chinese population.