The atmospheric pressure plasma at room temperature (ARTP) technology was applied to treat spores of Actinosynnema pretiosum FRCE-025. A high ansamitocin AP-3 producing strain ARTP-054 was obtained and the ansamitocin concentration reached 235.16 mg/L, which was increased by 43.3% compared with the original strain (164.12 mg/L). Chemical mutagenesis was further carried out on strain ARTP-054. The spores suspension was treated by 0.4 g/L nitrosoguanidine, and isobutanol, the branched chain precursor of ansamitocin AP -3, was used for substrate restrictive screening. Combined with the second-round screening, another highly producing strain HF-064 was finally screened and the yield of ansamitocin reached 287.64 mg/L, which was increased by 75.3% compared with the original strain. The 50 L fermentor test showed that the genetic stability of the mutant strain is good, and the yield of ansamitocin reached 292.36 mg/L after 168 h of fermentation.