Cytosine-substituted mildiomycin analogue (MIL-C) was synthesized by supplementing cytosine into the culture medium of Streptoverticillium rimofaciens (one mildiomycin producer) and had a specific and strong inhibitory activity against powdery mildews. In order to facilitate the separation of MIL-C from this fermentation broth, the cultivation conditions were optimized to increase MIL-C productivity, and more than 0.9g/l cytosine was added to the medium to inhibit the biosynthesis of mildiomycin (MIL). According to the ion-exchange equilibrium and dynamics characteristic of MIL-C, one weakly cationic resin (DK110) was screened out to separate MIL-C from fermentation broth with one effective ion-exchange method. When 2% ammonia aqueous solution was applied as eluent with 2 BVs/h, high recovery yield (97.6%) and purity (70.0%) of MIL-C were achieved. This novel strategy of combining up-stream biosynthetic controls and down-stream purification is very promising for efficient production of this novel nucleoside antibiotic (MIL-C).