The dietary energy level can affect ruminal microbiota, and further can affect rumen fermentation and fatty acid (FA) synthesis. In this study, we investigated the correlations between rumen bacteria and rumen fermentation parameters and intramuscular fat (IMF) FA profiles of Holstein bulls fed different energy diets via using 16S rRNA high-throughput sequencing and gas chromatography. The results showed that the improved dietary energy increased propionate, isobutyrate and isovalerate concentrations, and decreased acetate concentration and the acetate/propionate ratio. Increased dietary energy improved beef IMF content and had no effects on cooking loss, Warner-Bratzler shear force, water holding capacity, or drip loss. Increase dietary energy also decreased C18:0, C18:1 trans, C22:0, C20:3n-3, C22:6n-3, and saturated fatty acids, and increased C18:1 cis-9, C18:2n-6 trans, and monounsaturated fatty acids. 16S rRNA high-throughput sequencing analysis revealed that dietary energy had no impact on alpha diversity or the relative abundance of most of the major phyla and genera in rumen. In all dietary treatment groups, the dominant microbial phyla were Bacteroidetes (54.91%) and Firmicutes (33.60%), and the major microbial genus was Prevotella_1 (21.75%). Improved dietary energy decreased the abundances of Firmicutes and Tenericutes and increased that of Proteobacteria at the phylum level, while decreasing those of RC9_gut_group, and increased Prevotellaceae_UCG-004, Phocaeicola, Acetitomaculum, Lachnoclostridium_1, Prevotellaceae_UCG-003, and Anaerovibrio at the genus level. Spearman correlation analysis showed high correlations between rumen bacteria and fermentation parameters/IMF FA profiles. Collectively, our data indicated that dietary energy affects the ruminal microbiota, and further affects ruminal fermentation and IMF FA composition.