β-Casein is the main component of cow's milk protein, with A1 and A2 β-casein being the most common. Of these, A1 β-casein hydrolysate produces BCM-7, which can cause lactose intolerance, while A2 β-casein milk is more gentle on the gut. However, there is limited research on the composition of rumen microbiota, metabolites, and host metabolites in different genotype cows using metagenomics and metabolomics. In this study, we used multi-omics analysis techniques to perform enrichment analysis of differential metabolites, identifying three key metabolic pathways in all three groups: Arachidonic acid metabolism and Tryptophan metabolism. The metabolites in these pathways exhibited unique metabolic characteristics within each group. We then used random forests and ROC to predict key metabolites in these pathways, identifying that the signature metabolites in the A2A2 group were predominantly anti-inflammatory substances, including 12-HETE, PGD2-4d, and Arachidonic Acid. The signature metabolites in the A1A2 group and A2A2 group were Indoleacetaldehyde. The AUC of these signature metabolites was greater than 0.85. Macrogenic linear discriminant analysis (LDA >
2.5) found that the microorganisms with greater contribution were concentrated in the A2A2 group. Compared with the other two groups, g_Bacteroides and g_Parabacteroides were mainly enriched in the A1A2 group. In group A2A2, g_Xanthomonas and g_Acetobacter are mainly enriched. Then, the key microorganisms in A1A2 group were identified by correlation analysis as g_Bacteroides and g_Parabacteroides. The key microorganisms in group A2A2 were g_Acetobacter, g_Xanthomonas and g_Mannheimia, which were consistent with the results of LEfSe analysis. These microorganisms mainly affect the degradation of fiber in the diet, host metabolism and the occurrence of inflammation. In conclusion, our results provide theoretical basis and data support for the study of dairy cows with different genotypes of β-casein, and help to determine the potential biological functions of different genotypes of casein in dairy products and their effects on human health.