Two experiments were conducted to evaluate the effects ofBacillus subtilis natto, which was initially isolated from fermented soybeans on milk production, rumen fermentation and ruminal microbiome in dairy cows. In Experiment 1, 36 early lactation Chinese Holstein dairy cows (56 ± 23 days in milk) were randomly assigned to three groups: Control, cows were fed total mixed ration (TMR); BSNLOW, TMR plus 0.5 × 1011colony-forming units (cfu) ofB. subtilis natto/cow per day; and BSNHIGH, TMR plus 1.0 × 1011cfu ofB. subtilis natto/cow per day. During the 70-day treatment period, daily milk production and daily milk composition were determined in individual cows. The results showed that supplementing dairy cows with 0.5 × 1011and 1.0 × 1011cfu ofB. subtilis nattolinearly increased (P< 0.01) milk production (25.2 and 26.4 kg/dayv. 23.0 kg/day), 4% fat-corrected milk (27.3 and 28.1 kg/dayv. 24.2 kg/day), energy-corrected milk (27.3 and 28.2 kg/dayv. 24.2 kg/day), as well as milk fat (1.01 and 1.03 kg/dayv. 0.88 kg/day), protein (0.77 and 0.82 kg/dayv. 0.69 kg/day) and lactose yield (1.16 and 1.22 kg/dayv. 1.06 kg/day) but decreased milk somatic cell counts (SCC) by 3.4% to 5.5% (P< 0.01) in BSNLOW and BSNHIGH treatments compared with Control. In Experiment 2, four rumen-cannulated dairy cows were fed the basal diet from 1 to 7 days (pre-trial period) and rumen samples were collected on days 6 and 7; the same cows then were fed 1.0 × 1011cfu/dayB. subtilis nattofrom days 8 to 21 (trial period) and rumen samples were collected on days 20 and 21.B. subtilis nattowas discontinued from days 22 to 28 (post-trial period) and rumen samples were collected on days 27 and 28. Compared with the pre- and post-periods, ruminal pH decreased by 2.7% to 3.0% during the trial period (P< 0.01), whereas ammonia nitrogen (NH3-N), total volatile fatty acids and molar proportion of propionate (P< 0.01) and valerate (P< 0.05) increased. Molar proportion of acetate decreased and the acetate to propionate ratio was lower (P< 0.01) during the trial period. However, no differences for 24-hin saccodry matter digestibility were detected among different periods (treatments) though NDF digestibility was reduced in the trial and post-trial periods (P< 0.01). Compared with pre-trial period, total ruminal bacteria, proteolytic and amylolytic bacteria in rumen enumerated by culture methods increased by 15.0%, 16.2% and 11.7%, respectively (P< 0.01) but protozoa decreased to 5.35 log10cfu/ml (P< 0.01) during the trial period. These results demonstrate thatB. subtilis nattoimproves milk production and milk components yield, decreases SCC and promotes the growth of total ruminal bacteria, proteolytic and amylolytic bacteria, which indicate thatB. subtilis nattohas potential to be applied as a probiotic for dairy cows.