Mildiomycin (MIL) is a novel nucleoside agro-antibiotic which shows a specific and strong inhibitory activity against fungi, especially, powdery mildews. Because of its low action dosage, excellent environmental compatibility and remarkably low toxicity to human and animals, MIL is regarded as a kind of green biological pesticides. In this paper, the recovery and purification of MIL from fermentation broth were investigated by ion-exchange separation technique. The ion-exchange properties of MIL on HZ110TM resin, including ion-exchange equilibrium and dynamics in column process, were discussed. Results showed that the equilibrium exchange capacity of MIL was 26.5 mg per gram of wet resin at optimal pH value of pH 7.0. Ion-exchange isotherms of MIL can be well correlated with Freundlich equation. The ion-exchange capacity increased with raising the temperature. In the dynamic column process, breakthrough capacity was 29.3 mg per gram of wet resin. According to the ratio of desorption and eluent concentration of MIL, 2% ammonia aqueous solution was chosen as the eluent. And the total recovery ratio of MIL in ion-exchange process was up to 94.5%, and the enriched factor was about 8.