Objective To analyze the effects of miR-181a-5p overexpression on metabolites in the small intestines of mice with subcutaneous oral cancer by detecting changes in metabolites and metabolic pathways. Methods Three groups were included in study: Control group, negative control and miR-181a-5p overexpression group. To establish a subcutaneous oral cancer model in mice, variously treated cell suspensions were subcutaneously injected into the upper right of the groin in female M-NSG severely immunodeficient mice. Changes in pathology and small intestinal tissues were assessed by HE staining. Changes in mouse body weight were also assessed. Tandem orbitrap mass spectrometry and ultrahigh performance liquid chromatography-tandem time-of-flight mass spectrometry, were used to examine metabolites in the small intestines. By pre-analyzing the original data and quality rating sample data, XCMS was able to assess which metabolites were different among the groups. To identify unique metabolic pathways, KEGG enrichment analysis was used. Results A total of 170 distinct metabolites were found in the small intestinal tissues of Control and NC groups. Choline metabolism, alanine, aspartate, and glutamate metabolism, GABA synaptic metabolism, glycerophospholipid metabolism, cAMP signaling route, cancer center carbon metabolism, and niacin and niacin amine metabolic pathways were important signaling pathways for metabolite enrichment. In the NC group, 16 distinct metabolites with VIP values larger than 2 were found in the small intestines compared with the OE group overexpressing miR-181a-5p. Glycerin phosphorylcholine, palmitic acid, 3-hydroxybutyl carnitine, and β-hydroxybutyric acid were among the metabolites that significantly varied. The primary enhanced metabolic pathway was the choline pathway. Conclusions Mouse small intestines underwent slight changes from subcutaneous oral cancer with the greatest effect on metabolites critical for energy metabolism. The choline metabolic pathway was the pathway that selected absolutely metabolites in mouse small intestines with subcutaneous grafts of oral cancer.