Abstract: This experiment aimed to study the relationship between sperm motility and seminal plasma carnitine in Simmental bull. Twenty-six semen samples of Simmental bulls were collected, 12 of which were viewed as abnormal group, and 14 of which were viewed as normal group according to their sperm motility. Ultra-high Performance Liquid Chromatography-Quadrupole-Time-Of-Flight Mass Spectrometry (UPLC-Q-TOF MS) technique was used for non-target determination of samples, the Ultra-high Performance Liquid Chromatography-Quadrupole-Trap Mass Spectrometry (UPLC-Q-Trap MS) technology was used for broad-target lipid determination, principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) were used for statistical analysis. The results of multivariate statistics showed that the metabolic profile of seminal plasma in the abnormal group changed obviously compared with the normal group. The carnitine with significant difference detected by the two methods was analyzed, in the abnormal group, the concentration of decanoyl-L-carnitine, L-palmitoylcarnitine, stearoylcarnitine, free carnitine, acetyl-carnitine, hydroxybutyryl-carnitine, 3-hydroxyoctanoyl carnitine, myristoyl-carnitine and decenoyl-carnitine were significantly higher than those in the normal group (P<0.05). These carnitine concentrations were negatively correlated with the sperm motility of Simmental bulls, which would provide new methods and ideas for studying the determination of bovine semen quality.