Although the toxicity of neonicotinoid insecticides has been demonstrated in several studies, the information on metabolism, behavior, and health risk remains limited and has raised concerns about its potential toxicity. Thus, in this study we assessed the effects of nitenpyram using different sublethal concentrations (one-third and one-tenth of the acute LC₅₀ values) on various developmental and metabolic parameters from gene expression regulation in Drosophila melanogaster (model system used worldwide in ecotoxicological studies). As a result, nitenpyram sublethal concentrations prolonged the developmental time for both pupation and eclosion. Additionally, nitenpyram sublethal concentrations significantly decreased the lifespan, pupation rate, eclosion rate, and production of eggs of D. melanogaster. Moreover, the mRNA expression of genes relevant for development and metabolism was significantly elevated after exposure. Mixed function oxidase enzymes (Cyp12d1), (Cyp9f2), and (Cyp4ae1), hemocyte proliferation (RyR), and immune response (IM4) genes were upregulated, whereas lifespan (Atg7), male mating behavior (Ple), female fertility (Ddc), and lipid metabolism (Sxe2) genes were downregulated. These findings support a solid basis for further research to determine the hazardous effects of nitenpyram on health and the environment.