The purpose is to elucidate factors related to negative results of anti- H. pylori antibody test in cases with gastric mucosal atrophy. A total of 859 individuals without past history of eradication therapy for H. pylori (545 males, 314 females; mean age 52.4 years) who underwent an upper GI endoscopy examination and serological test were enrolled as subjects. Serological testing was performed using SphereLight H. pylori antibody J^®, and endoscopic findings of gastric mucosal atrophy by the classification of Kimura and Takemoto and post-eradication findings were analyzed. The positive rates for the anti- H. pylori antibody test in subjects with and without gastric mucosal atrophy were 85.6% and 0.9%, respectively. In analysis of subjects with gastric mucosal atrophy, a low positive rate and serum titer was observed in subjects with C1, C2 and O3 atrophy. When the analysis was performed separately in male and female subjects, low positive rate was observed in males with O3 atrophy and females with C2 atrophy. Suspected post-eradication endoscopic findings were more frequently observed in cases with C2 atrophy. In conclusion, negative result of anti- H. pylori antibody test was frequently observed in middle-aged subjects with C1, C2 and O3 gastric mucosal atrophy.