Abstract Bowman‐Birk inhibitor (BBI) is toxic when fed to certain insects, including the fruit fly, Drosophila melanogaster . Dietary BBI has been demonstrated to slow growth and increase insect mortality by inhibiting the digestive enzymes trypsin and chymotrypsin, resulting in a reduced supply of amino acids. In mammals, BBI influences cellular energy metabolism. Therefore, we tested the hypothesis that dietary BBI affects energy‐associated pathways in the D. melanogaster midgut. Through microarray and metabolomic analyses, we show that dietary BBI affects energy utilization pathways in the midgut cells of D. melanogaster . In addition, ultrastructure studies indicate that microvilli are significantly shortened in BBI‐fed larvae. These data provide further insights into the complex cellular response of insects to dietary protease inhibitors.