The juvenile hormone binding protein (JHBP) plays a key role in the protection and transport of the hormone to target tissues. In this report the sequence of the jhbp promoter comprising about 2000 bp is characterized. Using a minimized false positive algorithm, six putative regulatory elements, Hunchback, Heat shock factor binding element, Ultrabithorax, Broad-Complex Z3, Elf-1 and Chorion factor 1/ultraspiracle (CF1/Usp) were found in the distal promoter of the jhbp gene. Proteins from nuclear extract of Galleria mellonella fat body form four specific complexes with probe containing TATA box, five complexes with Inr probe and one protein complex with DPE probe. EMSA and footprinting analyses showed that one of the three CF1/Usp elements (starting at − 1053) has an exceptionally high affinity to Usp protein. An unknown, high-affinity Usp/EcRDBD-binding element (TCAACA-AAC-TGTTCA), distinct from 20-hydroxyecdysone response elements, was identified in the jhbp gene promoter, based on a footprinting assay. Deletions of jhbp promoter in the regions containing the CF1/Usp elements enhance the transcriptional activity of luciferase reporter gene in the Trichoplusia ni High Five cell line. Obtained data suggest that jhbp promoter is TATA- and Inr-driven, CF1/Usp elements exhibit inhibitory effect on jhbp expression, and an interaction between Usp and DNA relies on recognition of the consensus sequence (GGGTCA) and on ionic interactions of several phosphate groups outside from this element.