Abstract: An experiment was conducted to investigate the effects of saikosaponin (SSA) on in vitro fermentation parameters and bacterial quantity. Four treatments consisting of supplemental SSA at 0, 0.25, 0.5, and 1.0 g/kg dry matter (DM) were combined with 0.5 g total mixed ration (TMR), 50 mL basal media and 25 mL rumen fluid obtained from ruminally cannulated, lactating Holstein dairy cows. The treatments were randomly assigned to 5 of 20 incubation bottles. Cumulative gas production (GP) was continuously monitored by an automated trace gas recording system (AGRS-III, Beijing) at 39℃ during the 48 h incubation. Relative content of the bacterial community in the in vitro culture fluid was analyzed by real-time quantitative PCR. The results revealed that SSA at 0.25 g/kg could increase dry matter degradability (DMD, P=0.08), total gas production (GP, P<0.05) and the concentration of acetate, propionate and total volatile fatty acids (TVFA) (P<0.05). Adding SSA at 0.5 and 1.0 g/kg level did not affect GP kinetics and DMD, while it increased the concentration of acetate and TVFA (P<0.05). The supplement of SSA increased the relative quantity of Ruminococcus albus and Streptococcus bovis (P<0.05), but decreased the content of Prevotella brevis and Ruminobacter amylophilus (P<0.05). Therefore, SSA supplementation improved gas production, increased VFA concentrations and the quantity of major microbial species in the in vitro culture fluid, which indicates that SSA may be beneficial for the manipulation of rumen microbial fermentation in vitro.