Optimization of the feeding process for tautomycin production by Streptomyces spiroverticillatus was performed using glucose and/or maleic anhydride. The feeding of glucose was based on the reducing sugar content (lower than 8 g/L) at a cultivation time of 40 h. After addition of 2% (w/v) glucose, the biomass increased from 21 to 28 g/L, and that of tautomycin from 572.06 to 837.6 mg/L. Moreover, 1723.1 mg/L of tautomycin (increased by 201.21%) was obtained by feeding 0.2% (w/v) maleic anhydride solution at a pH between 4 and 7 in the broth. For the experiments in the 15 L fermentor, tautomycin content reached its highest level (1714.7 mg/L), which was 199.7% higher than that of control by feeding both glucose and maleic anhydride.