Heat shock protein 90 (HSP90), a molecular chaperone in cell, which can regulate multiple signaling pathways, plays a key role in the process of cell differentiation, development and transportation. To explore the role of the promoter in regulating the expression of heat shock protein 90, based on the HSP90 gene cDNA of Haliotis diversicolor from our lab, its 5' flanking region was cloned by genome walking and Tail-PCR techniques. Results showed that there is an 809 bp intron between translation initiation site (ATG) and first exon (94 bp). The length of 5' flanking region is 2800 bp before the first exon and 2811 bp before the predicted transcriptional start site (A). A TATA-box was located in the upstream -30 bp of the transcriptional start site (A). Potential transcription factor binding sites include ATF, TBP, Sp1, Oct 1, C/EBPalpha, NF-1, NF-kappaB, GATA-1, and Sox-2, etc. A CpG island was found by the CpG island prediction software, whose length is 131 bp. Eight firefly-luciferase reporter gene vectors with different deletions of HdHSP90 gene were constructed and transiently transfected into 293T cells, and the activity of dual-luciferase reporter gene was detected. The results showed that the core promoter is located between -98 to 83 bp, and the three transcription factors between -624 to -539 bp, Oct-1, C/EBPalpha, and NF-1, can inhibit the gene transcription.