In the present study, three primiparous lactating Holstein cows (260–285 d in lactation) were used in a 3 × 3 Latin square design to assess the effects of three doses (0·0, 0·4 and 0·8 μg/kg body weight) of lipopolysaccharide (LPS, Escherichia coli 0111:B4) on changes in ruminal microbiota and ruminal fermentation. Ruminal pH was linearly decreased (P< 0·001) by LPS challenge, and the concentrations of acetate, propionate, butyrate, total volatile fatty acids and amino N increased linearly (P< 0·001) according to the LPS dose. LPS infusion linearly decreased (P< 0·001) the organic matter degradability of alfalfa hay and soyabean meal in the rumen, but did not affect (P>0·10) the gene expression of Na+/K+-ATPase and monocarboxylic acid transporter-1, -2 and -4. A plot of principal coordinate analysis based on unweighted UniFrac values and analysis of molecular variance revealed that the structure of ruminal bacterial communities in the control was distinct from that of the ruminal microbiota in the cattle exposed to LPS. At the phylum level, when compared with the control group, LPS infusion in the tested cows linearly increased (P< 0·05) the abundance of Firmicutes, and linearly decreased (P< 0·05) the percentage of Bacteroidetes, Tenericutes, Spirochaetes, Chlorobi and Lentisphaerae. To our knowledge, this is the first study to report that intravenously LPS challenge altered the ruminal bacterial microbiota and fermentation profiles. The present data suggest that systemic LPS could alter ruminal environment and ruminal microbiota composition, leading to a general decrease in fermentative activity.