Milbemycins are used commercially as insect repellents and acaricides; however, their high cost remains a significant challenge to commercial production. Hence, improving the titer of milbemycins for commercial application is an urgent priority. The present study aimed to effectively increase the titer of milbemycins using a combination of genome re-sequencing and metabolic engineering. First, 133 mutation sites were identified by genome re-sequencing in the mutagenized high-yielding strain BC04. Among them, three modifiable candidate genes (sbi_04868 encoding citrate synthase, sbi_06921 and sbi_06922 encoding alpha and beta subunits of acetyl-CoA carboxylase, and sbi_04683 encoding carbon uptake system gluconate transporter) related to primary metabolism were screened and identified. Next, the DNase-deactivated Cpf1-based integrative CRISPRi system was used in S. bingchenggensis to downregulate the transcription level of gene sbi_04868. Then, overexpression of the potential targets sbi_06921-06922 and sbi_04683 further facilitated milbemycin biosynthesis. Finally, those candidate genes were engineered to produce strains with combinatorial downregulation and overexpression, which resulted in the titer of milbemycin A3/A4 increased by 27.6% to 3164.5 mg/L. Our research not only identified three genes in S. bingchenggensis that are closely related to the production of milbemycins, but also offered an efficient engineering strategy to improve the titer of milbemycins using genome re-sequencing.