This experiment was conducted to determine the effect of increasing the dietary levels of nattokinase (NK) addi-tives on milk production and composition, and blood metabolites in dairy cows. NK additives with high fibrinolytic activity were produced by the strain with similarity to the Bacillus amyloliquefacines. A total 20 mid-lactating Holstein cows (average body weight 652.00 ± 4.38 kg; average 2.25 ± 0.35 parity; average milk yield 24.4 ± 0.38 kg/d) were randomly assigned to three treatments (5 animals per treatment). Cows were fed TMR supplemented with DDGS 100 g for control, NK additives 100 g and 200 g for T1 and T2 treatment, respectively for 4 weeks. By the increases of NK additives, milk yield increased for T1 (1.35 kg/d) and T2 (0.98 kg/d) at the final period than at the initial period (p < 0.05). But milk protein content decreased for T1 (3.20%) and T2 (3.24%) than for control (3.56%) (p < 0.05). Blood triglyceride, T-CHO and BUN levels in T1 decreased compared with that in control and T2 (p < 0.05). This finding showed that 100g of NK additives could possibly have a positive effect in lactation performance of mid-lactation Holstein cows by increasing milk yield, improving protein metabolism and decreasing cholesterol in blood.