This study was conducted to investigate the effect of Soybean Peptides (SPs) on ruminal fermentation and microbial diversity in vitro.Four levels of SPs supplements(0,0.25%,0.50% and 0.75% dry matter basis)were tested.pH,NH3-N,Neutral Detergent Fiber (NDF), Acid Detergent Fiber (ADF), Dry Matter Digestibility (DMD) and the Total Volatile Fatty Acid (TVFA) were measured at 6,24,48 h of in vitro mixed incubation the fluids.Microbial populations were determined at 24 h and Microbial Proteins (MCP)were determined at 24 and 48 h.The gas production after 48 h in vitro incubation presented linear growth(p<0.05)and the highest content was the level of 0.5% SPs (dry matter basis). NH3-N concentration reached the highest concentration with 0.75% (dry matter basis) at 48 h. pH linearly increased (linear, p<0.05) from 6 to 48 h. The digestion of DMD increased with increasing doses of SPs at 24 h. NDF and ADF linearly (p<0.01) decreased by adding SPs. The concentration of TVFA linearly increased (p<0.05) at 6, 24 and 48 h, and reached the highest concentration at the level of 0.75% (dry matter basis). SPs decreased (p<0.05) the molar proportion of acetate and propionate, respectively at 24 and 48 h. Acetat to Propionate ratio (A/P; linear, p<0.05) increased at 48 h, and reached the greatest value at the level of 0.75% (dry matter basis) at 48 h.The populations of rumen Ruminococcus flavefaciens (R. flavefaciens), Ruminococcusalbus (R. albus), Fibrobacter succinogenes (F. succinogenes), Butyrivibrio fibrisolvens (B. fibrisolvens), Streptococcus bovis (S.bovis), Ruminobacter amylophilus (R. amylophilus) and Succinimonas amylolytica (S. amylolytica) were analyzed based on the total rumen bacterial 16S ribosomal deoxyribonucleic acid (rDNA). The relative abundance of R.flavefaciens and R.albus increased at 24 h,but the relative abundance of F.succinogenes decreased at this time. The incubation of SPs had no effect on the abundance of S.bovis and R.amylophilus.The relative abundance of B.fibrisolvens and S.amylolytica reached the greatest value(p<0.05)at middle doses of SPs inclusion at 24 h.The value of MCPs linearly increased (p<0.05)at 24 and 48 h.These results showed that SPs could improve in vitro fermentation and nutrient digestion of feed substrates, and had the ability to modulate the ruminal fermentation patt by regulating the composition of functional rumen microbes. Hence, SPs might be a potential feed additive applied in the diets of ruminants.