The botanical insecticide azadirachtin affects a variety of biological processes. Our early work indicated that protein level and type are significantly influenced by azadirachtin in pupae of Ostrinia furnacalis (Guenée) (Lepidoptera: Crambidae) because a correlation exists between protein content and azadirachtin concentration. By use of proteomic techniques, we analyzed changes in hemolymph protein expression of 48-h-old pupae in O. furnacalis induced by azadirachtin treatment. After feeding by third instars on an artificial diet containing 10 ppm azadirachtin until pupation, 48-h-old pupae were collected, and hemolymph protein samples were prepared. They were separated by two-dimensional polyacrylamide gel electrophoresis, and six proteins were significantly affected by azadirachtin treatment compared with an untreated control. Two of these proteins were identified by database searching with peptide mass fingerprinting by using matrix-assisted laser desorption/time-of-flight mass spectrometry after in-gel trypsin digestion. They belong to the insect apolipophorin-III and phosphoribosyltransferase family, respectively. These two proteins may function on lipid metabolism in insect hemolymph. Furthermore, fat body is the center of synthesis and secretion of hemolymph proteins. We suggest that the azadirachtin exerts its insecticidal effects on the fat body of O. furnacalis by interfering with protein expression related to hemolymph lipid metabolism.