<jats:title>ABSTRACT</jats:title> <jats:p> Avermectin (AVM) and ivermectin (IVM) are potent pesticides and acaricides which have been widely used during the past 30 years. As insect resistance to AVM and IVM is greatly increasing, alternatives are urgently needed. Here, we report two novel AVM derivatives, tenvermectin A (TVM A) and TVM B, which are considered a potential new generation of agricultural and veterinary drugs. The molecules of the TVMs were designed based on structure and pharmacological property comparisons among AVM, IVM, and milbemycin (MBM). To produce TVMs, a genetically engineered strain, MHJ1011, was constructed from <jats:named-content content-type="genus-species">Streptomyces avermitilis</jats:named-content> G8-17, an AVM industrial strain. In MHJ1011, the native <jats:italic>aveA1</jats:italic> gene was seamlessly replaced with <jats:italic>milA1</jats:italic> from <jats:named-content content-type="genus-species">Streptomyces hygroscopicus</jats:named-content> . The total titer of the two TVMs produced by MHJ1011 reached 3,400 mg/liter. Insecticidal tests proved that TVM had enhanced activities against <jats:named-content content-type="genus-species">Tetranychus cinnabarinus</jats:named-content> and <jats:named-content content-type="genus-species">Bursaphelenchus xylophilus</jats:named-content> , as desired. This study provides a typical example of exploration for novel active compounds through a new method of polyketide synthase (PKS) reassembly for gene replacement. The results of the insecticidal tests may be of use in elucidating the structure-activity relationship of AVMs and MBMs.