The aim of present study was to evaluate the association of common polymorphisms detected in mitochondrial DNA (mtDNA) D-loop region (mononucleotide repetitive D310, single nucleotide polymorphism (SNP) D16521) with susceptibility to gastric cancer (GC) in northwestern Chinese population. A total of 180 GC patients and 218 healthy controls were investigated by using PCR- denaturing high performance liquid chromatography (DHPLC) assay. Genotype and allele distributions and haplotype construction were analyzed in case-control study. We found D310 and D16521 heteroplasmy were significantly different between GC cases and controls (p < 0.05), and D16521 homoplasmy showed association with histological grade of GC (p < 0.05). Haplotype 7C/T, 8C/C and 9C/C had significant association with GC risk implied from analysis of D310 and D16521. Taken together, these findings suggested that mtDNA D-Loop polymorphisms and haplotypes may contribute to genetic susceptibility to GC in Chinese population.