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Background: Artificial reduction of internal ecdysteroid titer is very difficult to achieve in insects. Results: Injection of Nomuraea rileyi ecdysteroid-22-oxidase (E22O) or forced expression of the E22O gene reduced ecdysteroid titer and manipulated embryogenesis, molting, metamorphosis, and diapause in a number of insects. Conclusion: E22O is the first versatile ecdysteroid titer-decreasing tool. Significance: E220 will be used to answer various ecdysteroid-associated developmental and physiological questions.

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