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ABSTRACTEmerging evidence shows that the human microbiota plays a larger role in disease progression and health than previously anticipated. Helicobacter pylori, the causative agent of gastric cancer and duodenal and gastric ulcers, was early associated with gastric disease, but it has also been proposed that the accompanying microbiota in Helicobacter pylori-infected individuals might affect disease progression and gastric cancer development. In this study, the composition of the transcriptionally active microbial community and H. pylorigene expression were determined using metatranscriptomic RNA sequencing of stomach biopsy specimens from individuals with different H. pyloriinfection statuses and premalignant tissue changes. The results show that H. pyloricompletely dominates the microbiota not only in infected individuals but also in most individuals classified as H. pyloriuninfected using conventional methods. Furthermore, H. pyloriabundance is positively correlated with the presence of Campylobacter, Deinococcus, and Sulfurospirillum. Finally, we quantified the expression of a large number of Helicobacter pylorigenes and found high expression of genes involved in pH regulation and nickel transport. Our study is the first to dissect the viable microbiota of the human stomach by metatranscriptomic analysis, and it shows that metatranscriptomic analysis of the gastric microbiota is feasible and can provide new insights into how bacteria respond in vivoto variations in the stomach microenvironment and at different stages of disease progression.