Diet-induced milk fat depression (MFD) is a condition marked by a reduction in milk fat yield achieved by altering dietary conditions including increasing unsaturated fatty acids and fermentability of carbohydrates. 2-Hydroxy-4-(methylthio) butanoate (HMTBa) is a methionine analog that has been observed to reduce diet-induced MFD in dairy cows. Our hypothesis was that the reduction in diet-induced MFD by HMTBa was due to changes in the rumen microbiota. To test this, 22 high-producing cannulated Holstein dairy cows were arranged in a randomized block design and assigned to either control of HMTBa supplementation (0.1% of diet DM). Cows were then exposed to 3 diets with either a low risk (32% NDF, no added oil; fed d 1 to 14), a moderate risk (29% NDF and 0.75% soybean oil; fed d 15 to 21), or a high-risk (29% NDF and 1.5% soybean oil; fed d 21 to 28) for diet induced MFD. Rumen samples were collected on d 0, 14, 21 and 28, DNA extracted, V1-V2 region of the 16S rRNA gene PCR amplified, sequenced on Illumina MiSeq platform, and subjected to bacterial diversity analysis using the QIIME pipeline. The α diversity estimates (species richness) and shannon diversity were decreased in control compared with HMTBa (P < 0.05). Bacterial community composition also differed between control and HMTBa based on both unweighted uniFrac (presence/absence) and weighted uniFrac (relative abundance of commonly detected bacteria) distances (P < 0.01). Within the HMTBa group, there were no differences between d 0 and d 14, 21 and 28; however, in the control group, d 0 samples were different (P < 0.05) from d 14, 21 and 28. Bacterial genera including Dialister, Meghasphaera, Lachnospira, and Sharpea were increased in control compared with HMTBa (P < 0.05). Interestingly, these genera were positively correlated with milk fat trans-10, cis-12 CLA and trans-10 C18:1, isomers associated with MFD. It can be concluded that diet-induced MFD is accompanied by significant alterations in the bacterial community and HMTBa supplementation reduced rumen microbial perturbations when increasing dietary risk factors.