Metformin is widely prescribed to treat type 2 diabetes. Diabetes patients treated with metformin have a decreased risk of cancers, including gastric cancer. Among the factors influencing digestive carcinogenesis, gut microbiota interactions have been intensively studied. Metformin exhibits direct antimicrobial activity toward Helicobacterpylori, which plays a crucial role in gastric carcinogenesis. Mice were infected with H. pylori and treated for 12 days with either metformin or phosphate-buffered saline (PBS) as a control. At the end of the treatment period, the mice were euthanized and cecal and intestinal contents and stool were collected. The gut microbiota of the three different digestive sites (stool, cecal, and intestinal contents) were characterized through 16S RNA gene sequencing. In mice infected with H. pylori, metformin significantly decreased alpha diversity indices and led to significant variation in the relative abundance of some bacterial taxa including Clostridium and Lactobacillus, which were directly inhibited by metformin in vitro. PICRUSt analysis suggested that metformin modifies functional pathway expression, including a decrease in nitrate reducing bacteria in the intestine. Metformin significantly changed the composition and predicted function of the gut microbiota of mice infected with H. pylori; these modifications could be implicated in digestive cancer prevention.