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Objective: The relationship between Helicobacter pylori and the intestinal microbiota has not yet been clearly demonstrated in children and adolescents. The present study aimed at evaluating how H. pylori infection could affect the intestinal microbiota in adolescents using genetic analysis., Design: Cross-sectional study., Setting and Participants: We included subjects from a longitudinal project involving H. pylori screening and treatment of junior high school third-grade students (aged 14 or 15 years) in Saga Prefecture. The study included a control group (n=79) and an H. pylori group (n=80) tested negative and positive for the anti- H. pylori antibody in the urine and H. pylori antigen in stool specimens, respectively., Interventions: The intestinal microbiota was evaluated in stool specimens using 16S rRNA gene/DNA/amplicon sequencing with next-generation sequencing., Primary and Secondary Outcome Measures: We assessed alpha and beta diversity, just as well as relative abundances within the bacterial composition at the genus level in both groups., Results: As shown by the alpha diversity of the 16S rRNA gene/DNA/amplicon sequence data, the control group exhibited lower microbial species richness with lower alpha diversity compared with the H. pylori group (p<0.001). The beta diversity of the intestinal microbiota profile also differed between the two groups (p<0.01). The relative abundance of the Prevotella genus was higher in the H. pylori group (p<0.01) concomitant with a gain in body mass index (BMI) in the H. pylori group (p<0.01) compared with the control group., Conclusion: H. pylori infection significantly affected the intestinal microbiota in Japanese adolescents. In addition, the prevalence of the Prevotella genus is concomitantly increased along with the BMI in H. pylori -infected students., Trial Registration Number: UMIN000028721., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)