The microbiota of animals’ digestive tract plays important roles in health, nutrient utilization, and performance. For ruminants, the digestive tract, including rumen and other gastro-intestinal regions, hosts a unique and diverse microbiome that aids feed fermentation, digestion, and absorption. We hypothesize that compositions of diets can influence microbiomes and further affect the production of VFA and the composition of bile acids in beef cattle. We used rectum microbiota as a baseline and examined the microbiome in rumen and jejunum by16S-seq, together with quantifications of VFAs, lactate, and bile acids under grass-feeding and grain-feeding. We found that microbial communities displayed significant differences in microbiome structure and VFA production. Moreover, increased VFAs in grain-fed group may trigger lactate-associated bacteria growth. Bile acids could promote the growth of bacteria that had abilities of secondary bile acids conversion. These results provided deep insights into differences in beef quality and bovine biology under different diets.