The present study was undertaken to clarify the mechanism regulating cuticle protein gene expression. Expression of BMWCP5 was strong at around pupation and weak at the mid-pupal stage in wing tissues of Bombyx mori. We analyzed the upstream region of the BMWCP5 gene using a transient reporter assay with a gene gun system to identify the regulatory elements responsible for its unique expression pattern. We identified two betaFTZ-F1 binding sites to be important cis-acting elements for the transcription activation of the luciferase reporter gene by an ecdysone pulse. Site-directed mutagenesis of these sites, followed by introduction into wing discs, significantly decreased the reporter activity. We also found that the regions carrying the binding sites for the ecdysone-responsive factor BR-C Z4 (BR-Z4) were responsible for the hormonal enhancement of the reporter gene activity in wing discs. Mutation of the BR-Z4 binding sites decreased the reporter activity. The nuclear proteins that bound to these betaFTZ-F1 and BR-Z4 sites were identified by an electrophoretic mobility shift assay (EMSA). The results demonstrate for the first time that the BR-Z4 isoform can bind to the upstream region of the cuticle protein gene, BMWCP5, and activate its expression. The results also suggest that the BMWCP5 transcription is primarily regulated by the ecdysone pulse through betaFTZ-F1, and the stage-specific enhancement is brought about through BR-Z4.