[Abstarct] Objective By detecting changes in metabolites and metabolic pathways in the small intestine of mice with subcutaneous transplantation of oral cancer, the effects of overexpression of miR-181a-5p on metabolites and metabolites in the small intestine of mice with subcutaneous transplantation of oral cancer were analyzed. Methodes Three groups existed in the experiment: the Control group, the Negative control group, and the Over expression of miR-181a-5p group in the experimental group. To construct a subcutaneous transplantation tumor mouse model of oral cancer, different groups of treated cell suspensions were subcutaneously injected into the right point and upper location of the groin of M-NSG severely immunodeficient female mice. The pathogenic changes in each group were identified while additionally following the changes in the mice's body weight and small intestinal tissues using HE staining. By using tandem Orbitrap mass spectrometry and ultra-high performance liquid chromatography-tandem time-of-flight mass spectrometry, the metabolites in the small intestine of mice in the NC group, OE group, and Control group have been detected. By pre-analyzing the original data and quality rating sample data, XCMS was able to assess which metabolites were different between the Control group and NC group and between NC group and OE group. To establish the unique metabolic pathways, KEGG enrichment analysis was used. Results A total of 170 distinct metabolites were found in the small intestinal tissues of the 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 are important signaling pathways for metabolite enrichment. In the NC group, 16 distinct metabolites with VIP values larger than 2 were found in the small intestine of mice compared to the OE group that overexpressed miR-181a-5p. Glycerin phosphoylcholine, palmitic acid, 3-hydroxybutyryl carnitine, -hydroxybutyric acid, etc. are example of the metabolites which significantly vary. The primary raised metabolism path is the one for choline. Conclusions Mice's small intestine suffered slight changes as a result of subcutaneous transplantation of oral cancer, with the greatest effect in the metabolites critical in energy metabolism. The choline metabolic path was the pathway that selected absolutely metabolites in the small intestine of mice with the subcutaneous grafts of oral cancer.