Using molecular approaches, a new member of the Bombyx mori small heat shock protein family was cloned and characterized. The isolated gene contains an open reading frame of 672 bp, encodes a polypeptide of 223 amino acid residues with a predicted molecular mass of 25.4 kDa, and is therefore named BmHSP25.4. The gene codes for a protein homologous to the previously characterized HSP20.4 and HSP19.9. Western blotting analysis revealed that BmHSP25.4 existed in the fifth-instar larva's fatty body and blood tissues. Immunohistochemistry assay also showed that BmHSP25.4 was located in the fifth-instar larva's fatty body. The results of above studies have indicated constitutive expression of BmHSP25.4 in fatty body, blood tissues, and Bm5 cells. Finally, we examined the effect of heat stress on localization of BmHSP25.4 using anti-BmHSP25.4 polyclonal antibody by immunofluorescence. Under normal conditions, BmHSP25.4 was mostly found in the cytoplasm. However, after heat treatment, most of BmHSP25.4 distributed in the cell membrane. After 3 h of recovery following the heat shock treatment, the localization of BmHSP25.4 was the same as that under normal conditions.