Abstract: 【Objective】 The aim of this study is to investigate the difference and change of plasma metabolites before and after 3,600 m speed race in Yili horses. 【Method】 According to the genetic pedigree provided by the equestrian club, 5(3♂2♀) unrelated, weight (384.40±34.18)kg, and age 2 speed raced-horses were selected.The horses were trained daily with equal intensity. On the 25 th day of the experimental stage, the 3,600 m speed race was performed 4 h after morning feeding. The horse completed the race at (11.75±0.87) m/s at average speed. The rate of heartbeat and the breathing were measured immediately before and after the race. At the same time, blood was collected, plasma samples were prepared, plasma mass spectrometry was collected, and metabolites were determined by non-target metabolomics method. 【Results】 Compared with the pre-competition heart rate (39.80±0.40) bpm and respiratory rate (14.00±1.80) bpm, the horse heart rate (80.80±7.70) bpm and respiratory rate (78.80±3.90) bpm were significantly increased (P<0.05). Compared with Yili horses plasma at pre-race, the citric acid cycle, metabolism of alanine, aspartic acid and glutamic acid, transformation between pentaccharide and glucuronic acid, metabolism of cystine and methionine, oxidative phosphorylation, fatty acid degradation and sheath lipid metabolism showed significant changes. On the basis of the LC/MS analysis of plasma metabolomics of Yili horse before and after the 3,600 m speed race, LC/MS metabolic profiles of plasma samples of Yili horse 3,600 m before and after the race were different. The differential metabolites involved in citric acid cycle, glycolysis system, fat metabolism, amino acid metabolism. 【Conclusion】 During the 3,600 m speed race, the Yili horse body presents anaerobic, aerobic and mixed oxygen metabolism characteristics show anaerobic, aerobic and mixed oxygen metabolism.