The invention discloses a mildiomycin biosynthesis gene cluster belonging to the biotechnology field. The sequence of the gene cluster is disclosed in SEQ ID NO: 1; the gene cluster comprises 16 genes, wherein the gene cluster comprises 11 structural genes: milA, milB, milC, milD, milE, milG, milH, milJ, milM, milN and milQ; the gene cluster comprises two regulator genes: milK and milO; the gene cluster comprises one resistance gene: milP; and the gene cluster comprises two other genes: milF and milI. The invention provides all genes relevant to mildiomycin biosynthesis and protein information and provides the basis for mildiomycin biosynthesis and genetic engineering. The mildiomycin biosynthesis gene cluster and the protein thereof of the invention can be widely applied to the fields of agriculture, industry and medicine.