Azadirachtin, a tetranortriterpenoid botanical insecticide, has varied sub-lethal effects against many insect pests, including antifeedant, repellent, and growth regulatory. Despite extensive studies of the mechanisms that underline these physiological effects, little attention has been given to multiple toxic effects of azadirachtin under a coherent concentration, and there is no definitive overarching consensus on its toxicity. Here, we investigated multiple sub-lethal effects induced by 4 mg L-1 of azadirachtin, which did not elicit antifeedant behavior in Drosophila melanogaster, on metrics of longevity, development, compound eyes and reproduction. Exposure to < 20 mg L-1 azadirachtin did not induce mortality, and 4 mg L-1 of azadirachtin could shorten lifespan, expression of detoxification genes and activities of related detoxification enzymes were higher. The lower activity of chitinase and higher content of chitin in fruit fly exposed to 4 mg L-1 azadirachtin could be important in developmental inhibition effects, and ovarian abnormalities and lower fecundity could have resulted from azadirachtin-mediated influences on juvenile hormone and ecdysone that disrupted the endocrine system. Caspase-3, head involution defective and reaper-dependent apoptosis genes may have been responsible for compound eye abnormalities in flies exposed to azadirachtin. Our findings provide important insights to the potential mechanisms of sub-lethal effects of azadirachtin.