Objective: This study aimed to elucidate the mechanisms underlying milk composition divergence between naks (female yaks) and Simmental cows (S-cows) by integrating longitudinal multi-omics analyses of gut microbiota and metabolomes.Methods: We determined the gut microbiota and metabolites of both species over a 54-day period (day 26 to 80 of lactation) of ten naks and ten S-cows. Gut microbiota dynamics were assessed via 16S rRNA sequencing, while serum and fecal metabolomes were profiled using ultra-high performance liquid chromatography–tandem mass spectrometry. Statistical analyses included Wilcoxon rank-sum tests, linear discriminant analysis effect size (linear discriminant analysis>2, p<0.05), and Spearman correlations (r>0.70).Results: Milk yield was lesser (0.53–0.91 vs. 2.07–3.88 kg/d) but concentrations of fat (5.63%–6.30% vs. 3.30%–3.74%), protein (5.66%–6.30% vs. 3.39%–3.74%), and conjugated linoleic acid (CLA) (1.74%–2.35% vs. 1.40%–1.75%) were greater (p<0.001) in nak than Scow milk. Species-specific microbial signatures emerged. In naks, the g-Family-XIIIAD3011-group and g-norank-Ruminococcaceae were correlated with bile acid metabolism and CLA synthesis via 13-hydroxyoctadecadienoic acid transport. Additionally, the naks gut had a greater concentration of 13-hydroxyoctadecadienoic acid, a precursor of CLA, which may be transported to mammary cells via phosphatidylcholine and converted to CLA under the catalysis of fatty acid desaturase2. S-cows harbored g-Succinivibrio and g-Eubacterium-ruminantium-group, which are linked to galactose utilization and mTORmediated amino acid allocation. Metabolomics revealed naks-enriched steroid biosynthesis and taurine pathways (false discovery rate<0.05), while S-cows exhibited a lactating network associated with greater milk yield.Conclusion: Host-specific gut microbiota mediated nutrient allocation trade-offs. Naks optimized lipid-rich milk through bile acid and CLA metabolic networks, whereas S-cows enhanced yield via microbial-galactose synergies. This research underscores the pivotal role of the gut microbiome in mediating milk composition and suggests that microbiome manipulation could be a promising strategy to enhance milk quality in ruminants.