Natamycin is a very attractive antifungal agent with wide applications in medical and food industries. In order to improve the productivity of natamycin, the effects of cultivation conditions were investigated with Streptomyces gilvosporeus LK-196 in the shake flasks and 30-L fermentors. The results showed that dissolved oxygen and shear force would affluence the biosynthesis of natamycin significantly. The high concentration of natamycin (2.03g/L) was achieved under the suitable culture conditions in the shake flask scale. Further investigations in 30-L fermentors showed that the optimal pH was controlled at 6.0 during the whole bioprocess, and the dissolved oxygen level should be more than 30% by adjusting the aeration and agitation rates for high production of natamycin. Under these optimal conditions the high concentration of natamycin (3.94g/L) was achieved with Str. gilvosporeus LK-196 in the 30-L fermentor. Finally, the high-level fermentation process was successfully scaled up to 1000-L fermentors and 18,000-L fermentors in the pilot plant.