<jats:p>Spinosad, a member of polyketide-derived macrolides produced in the actinomycete <i>Saccharopolyspora spinosa</i>, has been developed as a broad-spectrum and effective insecticide. The β-oxidation pathway could be an important source of building blocks for the biosynthesis of spinosad, thus the effect of vegetable oils on the production of spinosad in a high-yield strain was investigated. The spinosad production increased significantly with the addition of strawberry seed oil (511.64 mg/L) and camellia oil (520.07 mg/L) compared to the control group without oil (285.76 mg/L) and soybean oil group (398.11 mg/L). It also revealed that the addition of oils would affect the expression of genes involved in fatty acid metabolism, precursor supply, and oxidative stress. The genetically engineered strain, in which <i>fadD1</i> and <i>fadE</i> genes of <i>Streptomyces coelicolor</i> were inserted, produced spinosad up to 784.72 mg/L in the medium containing camellia oil, while a higher spinosad production level (843.40 mg/L) was detected with the addition of 0.01 mM of thiourea.