Ten ruminally cannulated Holstein cows were used in a crossover design that investigated changes in ruminal bacterial populations in response to induction and recovery from diet-induced milk fat depression (MFD). Further, the effect on the ruminal microbiota of the cows with diet-induced milk fat depression inoculated with rumen contents from non-milk fat-depressed donor cows was evaluated. Milk fat depression was induced during the first 10 d of each period by feeding a low-fiber, high-starch, and high-polyunsaturated fatty acid diet (26.1% neutral detergent fiber, 28.1% starch, 5.8% total fatty acids, and 1.9% C18:2), resulting in a 30% decrease in milk fat yield. Induction was followed by a recovery phase, where all cows were switched to a high-fiber, low-starch, and low-polyunsaturated fatty acid diet (31.8% neutral detergent fiber, 23% starch, 4.2% total fatty acids, and 1.2% C18:2) and were allocated to (1) control (no inoculation) or (2) ruminal inoculation with donor cow digesta (8 kg/d for 6 d). Ruminal samples were collected at the end of induction (d 10) and during recovery (d 13, 16, and 28), separated to solid and liquid fractions, extracted for DNA, PCR- amplified for the V1-V2 region of the 16S rRNA gene, and analyzed for bacterial diversity. Results indicated that bacterial communities were different between fractions. In each fraction, differences were significant between the induction (d 10) and recovery (d 13, 16, and 28) periods; however, differences were less apparent with time during the recovery period. The MFD (d 10) was typified by a reduction in the relative sequence abundance of Bacteroidetes and an increase in the relative sequence abundance of Firmicutes and Actinobacteria across both fractions. At the genus level, relative sequence abundance of unclassified Lachnospiraceae, Butyrivibrio, Bulleidia, and Coriobacteriaceae were higher on d 10 and were positively correlated with trans-10,cis-12 CLA and the trans-10 isomer, suggesting their potential role in altered biohydrogenation reactions. A switch to the recovery diet resulted in a sharp increase in the Bacteroidetes lineages and a decrease in Firmicutes members on d 13; however, this shift appears to stabilize by d 28, indicating the restoration process for ruminal bacteria from an altered state is gradual and complex. Inoculation of 10% of rumen contents from non-MFD donor cows to MFD cows revealed this procedure had transient effects on only a few bacterial populations, and such effects disappeared after d 16 following cessation of inoculation. It can be concluded that alterations in milk FA profiles at induction are preceded by microbial alterations in the rumen driven by dietary changes.