Activated protein 4 (AP-4), an E-box DNA-binding protein, was cloned from the cotton bollworm, Helicoverpa armigera (Har). The expression of Har-AP-4 mRNA and the protein that it encodes are significantly higher in nondiapause pupae than in diapause pupae. In vitro-translated Har-AP-4 can bind specifically to the E-box motif on the promoter of the diapause hormone and pheromone biosynthesis-activating neuropeptide (DH-PBAN). Har-AP-4, fused with the green fluorescent protein (GFP), is localized to the nucleus, and overexpression of Har-AP-4 can significantly activate the promoter of the DH-PBAN gene that is involved in nondiapause pupal development in H. armigera. These results suggest that Har-AP-4, which binds to the promoter of DH-PBAN, may play a role in regulating pupal development in H. armigera.