The objective of this study was to evaluate the effects of lipopolysaccharide (LPS) dosing on ruminal bacterial community compositions (BCC). Diets were randomly assigned to 6 fermentors in a duplicated 3 × 3 Latin square with three 11-d experimental periods that consisted of 7 d for diet adaptation and 4 d for sample collection. Treatments were control diet (CON); wheat and barley diet (WBD), to induce subacute ruminal acidosis (SARA); and control diet + LPS (LPSD). Fermentors were fed 72 g of DM/d. The forage:concentrate ratio of CON was 65:35. The WBD was achieved by replacing 40% of the DMI of the CON diet with equal amount of ground wheat and barley; the LPS concentration in LPSD was 200,000 EU, which was similar to that observed in cows with SARA. The SARA inducing and LPS dosing started at d 8. The ruminal bacterial samples were collected at 0, 2, 5, and 9 h after morning feeding in both solid and liquid fractions. The bacterial community was determined by sequencing the V4 region of the 16S rRNA gene using the Illumina MiSeq platform. Ruminal pH of WBD was lower than LPSD and CON (P = 0.05), and WBD was able to keep ruminal pH below 5.6 for 240 min every sampling day, successfully inducing SARA. Ruminal pH of LPSD was not different from CON, and both were above 6 for the entire experimental period. The ruminal BCC of LPSD was different from WBD and tended to differ from CON in the solid fraction. In the liquid fraction, the ruminal BCC was different among treatments. Greater Bacteroidetes and lower Firmicutes were observed in WBD, while no difference was found between CON and LPSD. LPS dosing increased the relative abundance of Succinimonas, Anaeroplasma, Succinivibrio, Succiniclasticum and Ruminobacter but decreased the relative abundance of Treponema, Coprococcus and some Prevotella species, which are the main gram-negative bacteria associated with starch digestion. In conclusion, LPS dosing changed ruminal BCC, especially in liquid fraction and affected the growth of gram-negative bacteria associated with starch digestion in a dual flow continuous culture system.