OBJECTIVE: To construct a 5-oxomilbemycin producing strain by knocking out milF gene in Streptomyces hygroscopicus HS023. METHODS: Plasmid pMSST-ΔmilF was constructed and introduced into milbemycin industrial strain Streptomyces hygroscopicus HS023, and milF mutant F2-18 was selected by PCR amplification. RESULTS: Fermentation experiments showed that no milbemycins was produced in F2-18, but 5-ketomilbemycin, the intermediate of milbemycin, was obviously accumulated, and the fermentation titer was enhanced. CONCLUSION: Genetically engineered strain can simplify the synthesis of milbemycin oxime and lepimectin chemical from milbemycin.