To further clarify the biosynthesis and regulation pathways of avermectins and to increase its production,a bacterial artificial chromosome (BAC) library was constructed using the BamHⅠ-digested genomic DNA of Streptomyces avermitilis. The Streptomyces avermitilis BAC library was consisted of 2 304 clones with an average insert size of 101 kb,empty clone rate of about 9%,and 25.9-fold coverage of the whole genome. The DNA phosphorothioation of the Streptomyces avermitilis genome made it very difficult to construct a BAC library. The successful construction of this BAC library will provide a reference for constructing BAC libraries of other microorganisms, especially those with DNA phosphorothioation.