NW-G01, produced by Streptomyces alboflavus 313, is a novel cyclic hexapeptide antibiotic with many potential applications, including antimicrobial activity and antitumor agents. This study developed a system for optimizing medium components in order to enhance NW-G01 production. In this study, Plackett-Burman design (PBD) was used to find the key ingredients of medium components, and then response surface methodology (RSM) was implemented to determine their optimal concentrations. The results of PBD revealed that the crucial ingredients related to the production of NW-G01 were (NH4)(2)SO4, peptone and CaCO3. A prediction model has been built in the experiments of central composite design and response surface methodology, and its validation has been further verified. The optimal medium composition was determined (g/L): corn starch 15, glucose 15, peptone 3.80, (NH4)(2)SO4 0.06, NaCl 1.5, CaCO3 1.30, MgSO4 center dot 7H(2)O 0.015, K2HPO4 center dot 3H(2)O 0.015, MnCl2 center dot 4H(2)O 0.015, FeSO4 center dot 7H(2)O 0.015, and ZnSO4 center dot 7H(2)O 0.015. Compared with NW-G01 production (5.707 mg/L) in non-optimized fermentation medium, the production of NW-G01 (15.564 mg/L) in optimized fermentation medium had a 2.73-fold increase.