An experiment was conducted to explore the effect of diet-induced milk fat depression (MFD) on the global profile of rumen microbiota. Eight ruminally cannulated cows were subjected to time-course induction of and recovery from diet-induced MFD in a replicated design with 3 periods of 21 d. Briefly, MFD induction followed control and recovery followed MFD. A high-fiber, low-oil diet was fed during the control and recovery periods, and a low-fiber, high-oil (LFHO) diet was fed during the induction period. Whole ruminal digesta samples were collected and subjected to high-throughput Illumina sequencing of the V3-V4 hypervariable regions of bacterial 16S rRNA gene. On average, 40,562 high-quality sequences were generated per sample. Taxonomic classification of sequences unraveled the presence of 15 different bacterial phyla across all samples, among which Bacteroidetes (46.96%), Firmicutes (35.95%), Proteobacteria (7.5%), Spirochaetes (2.64%), and Fibrobacteres (2.10%) were identified as predominant members of rumen microbiota. When compared with control and recovery-associated communities, the diversity of rumen microbiota was significantly decreased following consumption of LFHO diet (P < 0.001). Moreover, PERMANOVA analysis of weighted UniFrac distances of microbial communities also revealed distinct clustering pattern for LFHO-associated microbiota compared with control and recovery diet (P = 0.002 and P = 0.007, respectively). Based on linear discriminant analysis effect size (LEfSe), the proportions of several members of rumen microbiota were also found to be significantly (P < 0.05) altered in response to dietary-induced MFD; the proportion of family Succinivibrionaceae (Proteobacteria) and genera Shuttleworthia and Catonella (Firmicutes) were found to be significantly increased in response to LFHO diet, while fibrolytic genera, such as Fibrobacter (Fibrobacteres) and Ruminococcus (Firmicutes) were found to be relatively more abundant during the control/recovery periods. Here, we demonstrated the complex dynamics of rumen microbiota that underlie diet-induced MFD.