Ethyl pyruvate (EP) has emerged as a promising compound with potential therapeutic benefits attributed to its anti-inflammatory and antioxidant properties. This study aimed to understand the effects of EP on plasma metabolites and immune cells in horses, utilizing advanced liquid chromatography-mass spectrometry (LC-MS)-based metabolomics, quantitative polymerase chain reaction (qPCR), and blood chemistry analyses. Our comprehensive analysis detected 2,366 ions, and 126 metabolites were accurately identified. Remarkably, EP administration induced significant changes in 28 metabolites at 1 h and 11 metabolites at 8 h, highlighting its time-dependent impact on metabolic pathways such as phenylalanine and arginine biosynthesis. Moreover, EP significantly lowered the expression of inflammatory markers interleukin (IL)-6 and heme oxygenase (HO)-1, indicating its potential as an anti-inflammatory agent. Blood chemistry analysis revealed notable reductions in glucose and triglyceride levels. These findings demonstrate that EP is a substance with potential effects on pathways associated with inflammation, oxidative stress, and metabolic processes.