Abstract BACKGROUND Several P450 genes in the 20‐hydroxyecdysone (20E) signaling pathway are known to critically regulate reproductive development in insects. However, the contribution of other P450 family members to insect reproduction remains poorly characterized. RESULTS Here, we identified a novel P450 gene, CYP12A2 , from the highly polyphagous pest Mythimna separata . Quantitative real‐time polymerase chain reaction analysis revealed that CYP12A2 was upregulated during sexual maturation and predominantly enriched in the ovaries of 3‐day‐old female adults. RNA interference (RNAi)‐mediated knockdown of CYP12A2 severely disrupted ovarian development and downregulated the expression of vitellogenesis‐related genes ( Vg and VgR ) as well as eggshell formation‐related genes ( CB‐Ld329 and FCP3C ), ultimately leading to reduced fecundity. Moreover, silencing of CYP12A2 significantly decreased 20E titers and suppressed the expression of key 20E biosynthetic genes ( CYP307A1 , CYP306A1 , CYP302A1 and CYP315A1 ). Importantly, exogenous 20E injection substantially rescued the defects in ovarian development and restored the expression of reproduction‐related genes that were impaired by CYP12A2 depletion. CONCLUSION These findings indicate that CYP12A2 regulates reproduction of M. separata by modulating 20E biosynthesis. Our study uncovers a novel reproductive role for CYP12A2 and highlights its potential as a target for RNAi‐based pest management strategies. © 2026 Society of Chemical Industry.
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