In modern breeding systems, cows are subjected to many stress factors. Animals fed with a high-grain diet may have a decreased rumen pH, which would lead to subacute ruminal acidosis syndrome. The aim of this study was to investigate the evolution of microbial community composition in cows undergoing a dietary stress challenge. Twelve cows were subjected to a challenge period consisted in a rapid change of ration, from a normal (45.4:54.6 forage: concentrate) to a high-grain content diet (24.8:75.2 forage: concentrate) to induce sub-acute ruminal acidosis. Individual rumen fluid content samples were collected before (T0), and during the challenge (T3, T14, T28). DNA from rumen contents was extracted, purified, and sequenced to evaluate Bacterial populations and sequencing was performed on Illumina MiSeq. The effect of animal conditions on rumen microbial community was quantified through a linear mixed model. The acidogenic diet created 2 main clusters: ruminal hypomotility (RH) and milk fat depression (MFD). The microbial composition did not differ in T0 between the 2 groups, while during the challenge Ruminococcus spp., Treponema spp., Methanobrevibacter spp., and Methanosphaera spp. concentrations increased in RH cows; Succinivibrio spp. and Butyrivibrio spp. concentrations increased in MFD cows. Prevotella spp. and Ruminococcus spp., were negatively correlated, while Christenellaceae family were positively correlated with both Methanobrevibacter spp. and Methanosphaera spp. Moreover, the same diet affected differently cows' microbiota composition, underlying the impact of the host effect. Other studies are necessary to deepen the relationship between microbiota composition and host.