A phytochemical study of corn silk resulted in the isolation of nine novel β-macrocarpene-type sesquiterpenoids ( 1– 9) along with one known analogue ( 10). The structural elucidation of these compounds was accomplished using comprehensive spectral data, X-ray crystallography, and electronic circular dichroism (ECD) calculations. Bioassay revealed that at a concentration of 20 μg/g, compounds 2, 3, 6, and 7 significantly reduced the weight of Spodoptera frugiperda, 4 inhibited the growth of S. litura, whereas compound 10 inhibited weight of both insects. In vitro enzyme assays showed that compound 10 inhibited carboxylesterase (CarE) and glutathione S-transferase (GST) enzyme activities in S. litura but did not impact these enzymes in S. frugiperda. Furthermore, transcriptome analysis suggested that 10 can upregulate the expression of the cytochrome P-450 monooxygenase (CPY450) and GST genes in S. frugiperda, whereas increased expression of carcinine transporter-like gene and decreased expression of trypsin were observed in S. litura. Overall, this research reveals the potential of corn sesquiterpenoids for developing environmentally friendly insecticides.