Within the 2.6-kb 5′ flanking region of the shrimp (Metapenaeus ensis) vitellogenin gene (MeVg2), several clusters of putative heat shock factor (HSF) response elements were identified. Deletion of these response elements has caused significant increases in MeVg2 promoter activity, suggesting that the HSF and Hsc70 complex may regulate vitellogenin gene expression in a negative manner. To confirm the role of Hsc70 in the regulation of vitellogenin gene expression, the ovary cDNA for Hsc70 was cloned and characterized. The Hsc70 transcript level was high in the ovary and hepatopancreas of females at the early vitellogenic stage but dropped during ovarian maturation. In addition, Western blot analysis revealed the presence of Hsc70 in the nuclear but not in the cytoplasmic fraction during the early stage of ovary maturation. Electrophoretic mobility shift assay (EMSA) results showed that ovary nuclear extract contained a factor that binds to the HSF response element. Since the addition of ATP caused a decrease in the binding of Hsc70, Hsc70 may form a repressor complex with HSF to inhibit MeVg2 expression. An in vitro RNA interference technique was used to study the gene function of Hsc70. Hsc70 gene knockdown resulted in an increased MeVg2 mRNA level in the ovary (54%) and hepatopancreas (62%). In summary, this report describes the first study of vitellogenin gene regulation at the transcription level in crustaceans and provides strong evidence that Hsc70 acts as a molecular chaperone to negatively regulate MeVg2 gene expression in shrimp.