Biopesticides are considered as an alternative to synthetic pesticide with a focus on increasing agricultural productivity as well as maintaining the ecosystem. Prior to application, its potential mechanism should be clearly addressed. Here, the effects of azadirachtin on the reproductive behavior in male Spodoptera litura (Fabricius) are determined. To further explore its molecular mechanism, an iTRAQ (isobaric tags for relative and absolute quantitation) based approach is applied to identify the differentially expressed proteins regulated by azadirachtin at two developmental stages. The results demonstrate that many proteins in the pathway of focal adhesion are regulated to exert influences in detachment of cell attachment, the loss of cell-cell interactions and inducing apoptosis at pupal stage, and many proteins in adenosine monophosphate-activated protein kinase pathway are also changed at the adult stage after azadirachtin-treatment as larvae. Moreover, based on their important roles, it is suggested that some proteins, such as ACTB-G1, ste20-related adaptor protein alpha, and regulatory-associated protein of mTOR (mTORC1) could serve as potential target proteins of azadirachtin to induce male infertility. The results of this study could provide evidence to illuminate the mechanism of male infertility induced by azadirachtin and potential targets for the development of environmentally friendly pesticides.