The yak (Bos grunniens) is a crucial species adapted to the extreme high-altitude environment of the Tibetan Plateau. However, a systematic understanding of how gut metabolic homeostasis contributes to this adaptation, particularly across altitudinal gradients and in comparison with sympatric species, remains limited. This study employed untargeted metabolomics to profile the intestinal contents of high-altitude yaks (HYs, 3800 m), low-altitude yaks (LYs, 2500 m), and plateau yellow cattle (PC, 3500 m) from the Gannan region. Multivariate analysis revealed distinct clustering of the three groups, with PLS-DA model validation parameters (R₂Y(cum) > 0.997, Q₂(cum) > 0.990), confirming significant species-specific and altitude-adaptive differentiation in the gut metabolome. A suite of differentially abundant metabolites (DAMs) was identified: HYs were enriched with stress- and inflammation-related molecules (e.g., prostaglandin E2, uric acid-indicative of oxidative stress and inflammatory responses) and steroid metabolites; LYs showed a profile oriented towards basic anabolism and antioxidant defense (e.g., L-galactono-1,4-lactone, allantoin); PC were characterized by unique lipid derivatives (e.g., 5-hexyl-2-furanhexanoic acid) and fungal-origin metabolites. Pathway enrichment analysis pinpointed Linoleic acid metabolism as one of the most significantly enriched pathways, with key hydroxyoctadecadienoic acids (HODEs) and related oxylipins significantly upregulated in HYs. Other significantly enriched pathways included Tryptophan metabolism, ABC transporters, and Protein digestion and absorption. Our findings suggest that high-altitude adaptation in yaks is associated with remodeling of the intestinal metabolic network. The identified signature metabolites and consistently enriched candidate pathways provide novel insights into potential host–microbiome-associated mechanisms and establish a metabolic foundation for the conservation of plateau livestock genetic resources and the development of precision husbandry strategies. However, these findings should be interpreted as hypothesis-generating because altitude, species, diet, and environment were not fully decoupled.