The role of genetics in the susceptibility to functional dyspepsia (FD) is not well established. Recently, two different associations were reported between FD and G-protein beta3 (GNB3) subunit gene polymorphism. We aim to clarify the association between GNB3 protein C825T polymorphism and dyspepsia in the Japanese population. Eight-nine dyspeptics and 94 nondyspeptic subjects enrolled in this study. All subjects underwent gastroscopy and patients with significant upper gastrointestinal findings were excluded. Other diseases were also excluded by face-to-face history and physical examination. GNB3 protein C825T polymorphisms were determined by polymerase chain reaction-restriction fragment-length polymorphism. H. pylori infection status was examined by histology or antibody against H. pylori. Nonsignificant correlation was found between GNB3 protein homozygous 825T and unexplained dyspepsia (OR = 1.65, 95% CI: 0.87-3.13). However, among H. pylori-negative subjects, homozygous GNB3 protein 825T significantly increased the risk of dyspepsia (16.7% versus 40.5%; CC versus TT; OR = 5.10, 95% CI: 1.21-21.43, CC versus others; OR = 3.40, 95% CI: 1.16-9.93, respectively). This significant association remained after logistic regression analysis with adjustment for sex and age (CC versus TT; OR = 5.73, 95% CI: 1.27-25.82, CC versus others; OR = 3.08, 95% CI: 1.02-9.25). No significant correlation was found between GNB3 polymorphism and any dyspeptic symptoms. Our data suggest that the homozygous 825T allele of GNB3 protein is associated with dyspepsia in the H. pylori-negative Japanese population. The role of genetics in the development of dyspepsia needs further evaluation.