Urokinase plasminogen activator (uPA) plays an important role in tumor invasion and certain inflammatory diseases. However, few studies have paid attention to how the uPA is associated with Helicobacter pylori infection and gastric atrophy. This study investigated associations of a C-to-T polymorphism of uPA (P141L, rs2227564) in exon 6 in 454 Japanese health checkup examinees (126 males and 328 females) aged 35 to 85 without a history of cancer. The uPA was genotyped by polymerase chain reaction with two-pair primers. The genotype distribution was in Hardy-Weinberg equilibrium (p=0.52) and the frequency of the T allele was 0.239. The risk of H. pylori sero-positivity was significantly reduced with the T/T genotype; the odds ratio (OR) relative to the C/C genotype was 0.34 (95% confidence interval [CI]: 0.14 to 0.86). Of the sero-negative subjects, 21 with atrophy were infected with H. pylori but lost their sero-positivity. After reclassifying them together with the sero-positive subjects, the corresponding OR was 0.40 (95% CI: 0.16 to 1.00), confirming that the T/T genotype decreased the risk of H. pylori infection. This gene polymorphism was not associated with the risk of gastric atrophy. In conclusion, this study indicated a possibility that the uPA minor homozygous genotype was associated with a reduction of H. pylori infection risk. Further studies are required to confirm these findings.