BackgroundDairy cows’ lactation performance is the outcome of the crosstalk between ruminal microbial metabolism and host metabolism. However, it is still unclear to what extent the rumen microbiome and its metabolites, as well as the host metabolism, contribute to regulating the milk protein yield (MPY).MethodsThe rumen fluid, serum and milk of 12 Holstein cows with the same diet (45% coarseness ratio), parity (2–3 fetuses) and lactation days (120–150 d) were used for the microbiome and metabolome analysis. Rumen metabolism (rumen metabolome) and host metabolism (blood and milk metabolome) were connected using a weighted gene co-expression network (WGCNA) and the structural equation model (SEM) analyses.ResultsTwo different ruminal enterotypes, with abundantPrevotellaandRuminococcus, were identified as type1 and type2. Of these, a higher MPY was found in cows with ruminal type2. Interestingly,[Ruminococcus] gauvreauiigroup andnorank_f_Ruminococcaceae(the differential bacteria) were the hub genera of the network. In addition, differential ruminal, serum and milk metabolome between enterotypes were identified, where the cows with type2 had higherL-tyrosine of rumen, ornithine andL-tryptophan of serum, and tetrahydroneopterin, palmitoyl-L-carnitine,S-lactoylglutathione of milk, which could provide more energy and substrate for MPY. Further, based on the identified modules of ruminal microbiome, as well as ruminal serum and milk metabolome using WGCNA, the SEM analysis indicated that the key ruminal microbial module1, which contains the hub genera of the network ([Ruminococcus] gauvreauiigroup andnorank_f_Ruminococcaceae) and high abundance of bacteria (PrevotellaandRuminococcus), could regulate the MPY by module7 of rumen, module2 of blood, and module7 of milk, which containedL-tyrosine andL-tryptophan. Therefore, in order to more clearly reveal the process of rumen bacterial regulation of MPY, we established the path of SEM based on theL-tyrosine,L-tryptophan and related components. The SEM based on the metabolites suggested that[Ruminococcus] gauvreauiigroup could inhibit the energy supply of serum tryptophan to MPY by milkS-lactoylglutathione, which could enhance pyruvate metabolism.Norank_f_Ruminococcaceaecould increase the ruminalL-tyrosine, which could provide the substrate for MPY.ConclusionOur results indicated that the represented enterotype genera ofPrevotellaandRuminococcus, and the hub genera of[Ruminococcus] gauvreauiigroup andnorank_f_Ruminococcaceaecould regulate milk protein synthesis by affecting the ruminalL-tyrosine andL-tryptophan. Moreover, the combined analysis of enterotype, WGCNA and SEM could be used to connect rumen microbial metabolism with host metabolism, which provides a fundamental understanding of the crosstalk between host and microorganisms in regulating the synthesis of milk composition.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40104-023-00862-z.