The objective of this study was to investigate the ruminal bacterial communities as affected by monensin, haylage, and their interaction of feedlot cattle fed 60 % dried distillers grains with solubles in a replicated 4 × 4 Latin square design. Pyrosequencing analysis of the V1-V3 region (about 500 bp) of 16S rRNA gene from the four dietary treatments (3 treatment plus one control diets) collectively revealed 51 genera of bacteria within 11 phyla. Firmicutes and Bacteroidetes were the first and the second most predominant phyla, respectively, irrespective of the dietary treatments. Monensin supplementation decreased the proportion of Gram-positive Firmicutes while increasing that of Gram-negative Bacteroidetes. However, the monensin supplementation did not reduce the proportion of all genera of Gram-positive bacteria placed within Firmicutes and lowered that of some genera of Gram-negative bacteria placed within Bacteroidetes. Haylage supplementation appeared to attenuate inhibition of monensin on some genera of bacteria. Factors other than monensin and haylage could affect ruminal bacterial communities.