Display omitted •The gut microbiome profile of a rat model of MNNG-induced gastric carcinogenesis was analyzed.•Changes in gut microbiota composition were detected by 16S rRNA gene sequencing.•Bacterial species richness increased and diversity decreased during gastric carcinogenesis progression.•The most significant changes were occurred at the precancerous lesion of GC stage.•Gut microbiome changes in the rat gastric carcinogenesis model were similar to those in human. Although many studies have examined changes in gut microbiota composition in gastric carcinogenesis to clarify the mechanism of action of anticancer drugs, it is unclear whether animal models of gastric carcinogenesis adequately reflect the disease in humans. To address this issue, the present study investigated changes in the gut microbiome profile of a rat model of gastric carcinogenesis established using a combination of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), sodium salicylate, irregular fasting, and ranitidine. The rats were divided into control (Normal), chronic non-atrophic gastritis (CNAG), chronic atrophic gastritis (CAG), precancerous lesion of gastric cancer (PLGC), and gastric cancer (GC) groups according to histopathological features. Gut microbiome in gastric carcinogenesis profiling was performed by 16S rRNA gene sequencing of rat feces samples. We found that gut bacterial species richness increased whereas species diversity decreased during gastric carcinogenesis, with the most significant changes detected in the PLGC group. Gut microbiota community composition differed across groups, with the greatest similarities observed between CNAG and CAG groups and between PLGC and GC groups. There were significant differences in taxonomic representation at the phylum level: the PLGC group had the highest ratio of Firmicutes/Bacteroidetes whereas the GC group had the highest abundance of Proteobacteria and Actinobacteria. These results indicate that changes in the gut microbiome in a rat model of MNNG-induced gastric carcinogenesis are similar to those observed in humans, thus providing a useful tool for evaluating the efficacy and mechanism of action of novel monotherapies or drug combinations for the treatment of gastric carcinogenesis.