Objective To analyze differences in the intestinal microbiota and transcriptomics between Nmethyl-N’-nitro-N-nitrosoguanidine (MNNG) gastric cancer rats and normal rats and to analyze the correlation between the two, so as to provide a reference for related studies using MNNG gastric cancer rats as a model. Methods A total of 12 Wistar rats were randomly divided into normal (NM) and gastric cancer (GC) groups. The GC group was given a concentration of 20 mg/mL of MNNG by gavage with a dose of 100 g/mL once a day, and the NM group was given the same amount of normal saline by gavage. Samples were collected for testing after 16 weeks of continuous intervention. The gastric tissues were collected and stained by HE staining to observe morphological changes in the gastric mucosa of the two groups, and the expression levels of differential genes were detected by transcriptome sequencing. The cecal contents were collected for 16S rRNA sequencing. Results (1) Visual observation and HE Results showed that the volume of gastric mucosa in the NM group was normal, the surface was glossy, the gastric wall was elastic, the direction of the mucosal folds was regular, there were no hyperplasia or hemorrhagic spots. In the GC group, the volume of gastric mucosa was reduced, the gastric wall was thinned, elasticity was poor, the direction of the folds was disordered and irregular, and there was a bulge accompanied by yellow-black keratotic hyperplasia. In the NM group, the squamous epithelial layer, submucosa, and muscular layer of the gastric mucosa were clear, with no hyperplasia and keratinization. In the GC group, the gastric mucosa had disorganized layers and cell polarity, with different cell morphologies; the squamous epithelial layer was destroyed, and squamous epithelial cells were hyperplasic, keratinized, and had invaded the muscular layer by proliferation. The modeling was considered successful. (2) The Results of intestinal microbiota sequencing showed that the abundance of Akkermansia and Lactobacillus in MNNG gastric cancer rats decreased significantly, and the abundance of the rumen coccaceae Prevonella, and Blauter increased significantly. (3) The three key pathways obtained by transcriptomic sequencing and KEGG pathway enrichment analysis were amebiasis, systemic lupus erythematosus, and the PI3K-Akt signaling pathway, and five genes differentially enriched in these three pathways were those for MCPT8I2, IGH-6, IGHG1, ACTN2, and VEGF-D. (4) Combined analysis of intestinal microbiota and transcriptomics showed that _UCG-005, Prevonella _UCG-003 and Brautella were positively correlated with amebiasis, systemic lupus erythematosus, and the PI3K-Akt signaling pathway. Conclusions The abundance of intestinal microbiota in gastric cancer rats formed by MNNG gavage is different from that of normal rats. The genes for MCPT8I2, IGH-6, IGHG1, ACTN2 and VEGF-D may be up-regulated in gastric cancer induced by MNNG gavage. Combined analysis of intestinal microbiota and differential genes suggested that the mechanism of MNNG carcinogenesis may be mainly related to the destruction of gastric mucosa and the inflammatory response.