Objective To investigate the changes of metabolic pathways in the process of immunesuppression in mice caused by cyclophosphamide by metabolomic analysis. Methods Flow cytometry was used to detect the changes of immune cells, and the results were analyzed using partial least squares model. The potential biomarkers were screened by variable importance in projection (VIP) parameters in the partial least squares model, and the differential metabolites were determined by statistical analysis. The differential metabolites were identified using the metabolomics rapid identification and analysis software. Nine kinds of metabolites were identified by searching and calculation, and result of pathway enrichment showed three differential metabolic pathways.Results Cyclophosphamide had a significant effect on the biosynthesis of unsaturated fatty acids, metabolism of mitochondrial fatty acids, metabolism of glycophospholipid, biosynthesis of steroid hormones, metabolism of arachidonic acid, metabolism of fatty acids, and the biosynthesis of pyrimidine.Conclusions The most important metabolic pathways affected by cyclophosphamide in the normal body are the metabolism of arachidonic acid, glycophospholipid and pyrimidine.