Echinacea purpurea is known as an important herbal medicine and has been frequently used for over last 300 years as a medicinal herb. This herb is well known for its effects on immune system, which can change the contents of different metabolites in animals. The mechanisms of its immunoregulation on various metabolomics are poorly known and are still unclear. Therefore, this study was executed to explore the effects of cyclophosphamide (CY) on normal control group (NC group) treated with sterile saline) and treatment group exposed to Echinacea extract (EE) to determine the effects on immunosuppression group (IS group, induced by CY) and its association with metabolomics. Here we reported that body weight, indices of immune organs, serum biochemical and cytokine contents were affected by CY and EE had the potential to reverse adverse effects through increasing IFN-γ and TNF-α level, significantly increased quantity of total protein (TP), and albumin (ALB). The results on the Gas Chromatography-Mass Spectrometer(GC-MS) technique illustrated that amino acid and energy metabolism involved in differential metabolites between IS and NC groups with vitamin B6 metabolism, pantothenate, and coenzyme A(CoA) biosynthesis metabolic pathways. Results indicated that myo-inositol, lactic acid, pyruvate are potential marker substances between EE-IS group and IS group with a principal impact on the metabolic pathways including inositol phosphate metabolism, phosphatidylinositol signaling system, ascorbate and aldarate metabolism and pyruvate metabolism. Overall, this study identified the involvement of different metabolites pathways of EE in immunosuppression rats which provided valuable evidence of EE immunoregulation effect via metabolomics and multiple bioanalytic methods.