The host microbiota is closely associated with tumor initiation and progression in multiple solid tumors including gastric cancer. The aim of this study was to investigate in patients with gastric cancer whether there are alterations in gastric microbiota and any potential association these may have with immune dysregulation. 16S rRNA gene sequencing was used to analyze tumor microbiota of 53 patients with gastric cancer and gastric mucosal tissue microbiota of 30 patients with chronic gastritis. The effect of microbiota on the tumor microenvironment (TME) was studied by single-cell sequencing, immunohistochemistry, multiplex immunofluorescence staining, and flow cytometry, as well as in a mouse model of primary gastric cancer. The gastric cancer microbiota was characterized by reduced microbial diversity and enrichment of the Oceanobacter, Methylobacterium, and Syntrophomonas genera. Intratumoral Methylobacterium was significantly associated with poor prognoses in patients with gastric cancer. It also was inversely correlated with the frequency of CD8+ tissue-resident memory T (TRM) cells in the TME. TGFβ was significantly reduced in gastric cancer samples with higher abundance of Methylobacterium. Finally, we verified that Methylobacterium can decrease TGFβ expression and CD8+ TRM cells in the tumor by establishing a mouse model of primary gastric cancer. The results suggest that tumor microbiota and exhausted CD8+ TRM cells in the TME of gastric cancer are significantly correlated, and that Methylobacterium may play a role in gastric carcinogenesis.