Abstract: The objective of this study was to examine the effects of alfalfa flavonoids on the bacterial flora in the rumen of dairy cows. Four primiparous Holstein cows fitted with ruminal cannulas were used in a 4×4 Latin square design and were fed a total mixed ration containing 0, 20, 60, or 100 g alfalfa flavonoids per day (group A-D, respectively). The experiment had four periods and each period lasted 24 days. The ruminal fluid was collected during each period. Total bacterial DNA was extracted from the ruminal fluid, and 16S RNA sequences were obtained using the Illumina MiSeq platform. In total, 52747 operational taxonomic units (OTUs) were generated, and 16142 OTUs were shared by four groups, accounting for 30.60% of the total OTUs. The largest amount of OTUs was in group A. As the amount of alfalfa flavonoids in the diet increased, the Shannon index first increased and then decreased, but the abundance-based coverage estimator and Chao 1 indexes were unaffected. Simpson’s index was significantly higher for group C than for groups A and D (P<0.05). There were more bacterial genera in group C than in groups B and A, but the number of phyla in each group was not affected by the amount of alfalfa flavonoids in the diet. At the phylum level, the relative abundance of SR1 tended to increase linearly (0.05<P<0.10), and the relative abundance of Synergistetes first decreased and then increased (0.05<P<0.10) as the amount of alfalfa flavonoids in the diet increased. At the genus level, the relative abundance of RC9 and SP3-e08 was higher in group B than in group A, while the relative abundance of Oribacterium was higher in group A than in group B. The relative abundance of Shuttleworthia increased linearly with increasing alfalfa flavonoids (P<0.05), and the relative abundance of Shuttleworthia was significantly higher in group D than in group A. In a clustering analysis, the bacterial community structure was similar in groups A and B, and in groups C and D. In conclusion, dietary supplementation with alfalfa flavonoids can change the composition and community structure of ruminal bacteria, thus affecting metabolism and nutrient digestion.