Abstract: The composition of seed culture medium and the culture conditions for cycloheximide-producing new strain Streptomyces yunnanensis YIM41004 were optimized.The optimum composition was as following:glucose 25g,peptone 7.5g,yeast extract 7.5g,CaCO3 3g,MgSO4·7H2O 1g,MnSO4·4H2O 0.1g,KH2PO4 0.2g,distilled water 1000mL.The optimal pH value was 7.8,the broth volume in 500mL flask was 100mL,and the optimal seed age was 32-36h.Inoculated with 10%(V/V) seed broth and cultured with the optimized seed culture medium,the cycloheximide production level of flask ferrmentation reached up to 498.9(mg·L-1),which was 10.7%higher than that cultured with the original seed broth culture.