Colonization of specific bacteria in the human mouth was reported to be associated with gastric cancer risk. However, previous studies were limited by retrospective study designs and low taxonomic resolutions. We performed a prospective case-control study nested within three cohorts to investigate the relationship between oral microbiome and gastric cancer risk. Shotgun metagenomic sequencing was employed to characterize the microbiome in prediagnostic buccal samples from 165 cases and 323 matched controls. Associations of overall microbial richness and abundance of microbial taxa, gene families and metabolic pathways with gastric cancer risk were evaluated via conditional logistic regression. Analyses were performed within each cohort, and results were combined by meta-analyses. We found that overall microbial richness was associated with decreased gastric cancer risk, with an odds ratio (OR) per standard deviation (SD) increase in Simpson's reciprocal index of 0.77 (95% confidence interval [CI] = 0.61-0.99). Nine taxa, 38 gene families and six pathways also showed associations with gastric cancer risk at P < .05. Neisseria mucosa and Prevotella pleuritidis were enriched, while Mycoplasma orale and Eubacterium yurii were depleted among cases with ORs and 95% CIs per SD increase in centered log-ratio transformed taxa abundance of 1.31 (1.03-1.67), 1.26 (1.00-1.57), 0.74 (0.59-0.94) and 0.80 (0.65-0.98), respectively. The top two gene families (P = 3.75 × 10^-4 and 3.91 × 10^-4 ) and pathways (P = 1.75 × 10^-3 and 1.53 × 10^-3 ) associated with gastric cancer were related to the decreased risk and are involved in hexitol metabolism. Our study supports the hypothesis that oral microbiota may play a role in gastric cancer etiology.