Seminal plasma (SP) is the product of testes, epididymis, and accessory sex glands released during ejaculation, making up 98% of the volume of the stallion ejaculate. Analyzing the entire metabolome allows to assess the final products of the biological system metabolism. The present study aimed to investigate the metabolomics of stallion SP. Twenty-four Criollo stallions with a known reproductive history and ≥30 inseminated mares were once collected during the breeding season. Pregnancy rates (day 16 after artificial insemination) ranged from 20.2% to 95.6%. Two groups were formed: High Pregnancy (HP; pregnancy rate per cycle ≥50%), and Low Pregnancy (LP; pregnancy rate of ≤49%). Microscopic semen analysis was performed using a computer-assisted sperm analysis system. Membrane physical integrity was assessed by fluorescent probes, and the hypoosmotic test analyzed membrane functional integrity. After centrifugation at 400xg for 10min centrifugation, SP plasma was transferred to a 2mL microcentrifuge tube and centrifuged again (10.000xg, 60min, 4°C). The supernatant was mixed 1:1 with deionized water after filtering with 0.45μm filters to remove cell sediment. Each sample was again filtered through a 0.22μm syringe filter before profile acquisition. The metabolic profile was acquired using liquid chromatography coupled with time-of-flight mass spectrometry. A total of 18 target metabolites were identified after analysis. The metabolites were tryptophan, lactic acid, ornithine, stearic acid, oleic acid, palmitic acid, melatonin, acetylcarnitine, carnitine,phenylalanine, isoleucine, isovaleryl, citric acid, hippuricacid, 1,3-dioxan-5-ol, taurine, phosphoric acid, and fructose. Groups were considered independent factors while the metabolite profile was considered a dependent factor. The Shapiro-Wilk Test was used to evaluate data distribution of SP metabolomics. The Kruskal Wallis test for nonparametric data and ANOVA and Tukey test for parametric data were used. Pearson's correlation was performed with a significance level of P<0.05. Four metabolites: oleic acid, isoleucine, taurine, and phosphoric acid, presented a difference (P<0.05) between the HP and LP groups. The oleic acid and isoleucine were higher in the LP group, while taurine and phosphoric acid were higher in stallions from the HP group. Phosphoric acid presented a relevant negative correlation with immobile spermatozoa (r=-0.7206) and a positive correlation with functional integrity (r=0.6162), physical integrity (r=0.6306), fertility (r=0.6471), progressive motility (r=0.5145), total motility (r=0.7157), VCL (r=0.6034), VSL (r=0.5491), VAP (r=0.5629), BCF (r=0.7055), ALH (r=0.5945), the total volume of the ejaculate (r=0.5941) and total protein/ejaculated (r=0.5182). Metabolites are products of cell signaling pathways and can be used as biological markers. Phosphoric acid contributes to the energy metabolism of sperm by breakdown of an inorganic pyrophosphate molecule. In conclusion, phosphoric acid in stallion SP should be considered as a marker of fertility.