The Bombyx mori silkworm's homologue of the Broad-Complex gene (BmBR-C) is transcribed from two promoters: a distal promoter (Pdist) and a proximal promoter (Pprox). As determined by a luciferase assay, the transcriptional activity of Pdist, but not Pprox, was activated by ecdysone. Further analyses using reporters driven by sequential deletion Pdist mutants indicated that two regions, ecdysone responsive element (EcRE)-D and EcRE-P, -4950 bp and -3480 bp upstream from the distal transcription start site, respectively, were important in the responsiveness of Pdist to 20-hydroxyecdysone (20E); however, no significant sequence similarities were found between the canonical EcRE and the EcRE-D or EcRE-P regions. Electrophoretic mobility shift assays showed that both the EcRE-D and -P sequences specifically bound to Bombyx protein(s). Sequence analyses and competition assays suggested that the protein(s) bound to EcRE-P might include components other than the ecdysone receptor (EcR), suggesting that BmBR-C transcription was indirectly activated by ecdysone through the EcRE-P. Remarkably, protein binding to the mid-region of the EcRE-D, EcRE-Db, was competitively inhibited by an oligonucleotide containing the Drosophila hsp27 EcRE sequence. Furthermore, an anti-EcR antibody interfered with the formation of the protein-EcRE-Db complex. These results indicated that a functional Bombyx ecdysone receptor binds to EcRE-D and activates the expression of BmBR-C.