Nattokinase is a novel fibrinolytic enzyme that is considered to be a promising agent for thrombosis therapy. In this study, Bacillus subtilis strain D was cultured in fed-batch fermentation for improving the production of nattokinase. In fed-batch culture, feeding was started after 5 hour of batch culture. The feeding strategies for nutrient were glucose supplying with 1 or 2 g/L/h and pH-stat using mixture of glucose and HCl, and only glucose. The nattokinase activity in fed-batch culture with pHstat using only glucose feeding was 4, 4.6, and 7 times higher than in fed-batch witp constant glucose feeding, pH-stat using mixture of glucose and HCl, and batch culture, respectively.