Abstract: The purpose of the present study was to examine the effect of different concentration of Bacillus subtilis natto on ruminal microbial fermentation in persistent artificial rumen system. Three groups, including control group (CK), treatment 1 group (TR1) supplied 1% B.subtilis natto of fermenter capacity and treatment 2 group (TR2) supplied 5% B.subtilis natto of fermenter capacity were employed. Results indicated that， compared with CK, pH values of TR1 and TR2 increased, but the difference was not significant (P>0.05). The NH₃-N concentration of TR2 was always higher than that of CK (P<0.05), while TR1 failed to increase NH₃-N concentration significantly (P>0.05). Compared with CK, the MCP concentration of TR1 and TR2 decreased significantly (P<0.05) and their concentration of acetate, propionate, butyrate, and total volatile fatty acids all increased to different extent. Compared with CK, the concentration of acetate, propionate, butyrate, and total volatile fatty acids in TR2 increased by 6.52%, 1.96%, 11.02% and 5.77% (P<0.05), respectively. TR2’s acetate/propionate ratio also increased significantly (P<0.05). However, no significant difference was observed between TR1 and TR2 (P>0.05) in all volatile fatty acids except that butyrate concentration was higher in TR2 than that in TR1 (P<0.05). The results suggested that B. subtilis natto has a negative effect on N metabolism, but could enhance the carbohydrate digestion by rumen microbes.