In order to study more about the molecular mechanism of nacre formation, we cloned a novel shell matrix protein hic9 (accession no. MF508725) from freshwater mussel Hyriopsis cumingii. We analyzed the gene expression by RT-PCR and in situ hybridization.The result indicated that hic9 was expressed most highly in adductor muscle and mantle, and the strong signals occurred in the whole outer epithelial cells of outer fold, and also occurred in the crust groove between out fold and middle fold. This result indicated that hic9 is a multifunctional matrix protein involved in the cuticle, prism layer and nacre layer of the shell. The amino acid sequence of hic9 featured high proportion of Gly (14.81%)、Pro (13.58%) and Ala (12.35%) residues, and formed the "Gly-X-X" structure in the middle of the sequence (X is any amino acid). And there is the continuous repetitive structure of alanine (polyA) structure close to the C terminal of the sequence, together giving the sequence of hic9 similar characters of spider silk protein. The Hic9 C terminus is composed of a hydrophobic sequence, "LAWMLFV", and this sequence is presumed to form a beta folding structure. Next to this sequence, the 89–91 bit is the "Asp-Leu-Asp" sequence, which is a typical Ca²⁺ binding site.In addition, the expression of hic9 in primary pearl sac was detected by real-time quantitative PCR in the early stage of pearl formation. Expression levels of hic9 in the pearl sac during 3–15 days after implantation maintained roughly the same, and the expression level of hic9 had a significant increase during 18–25 days in the period of transition from irregular to orderly in calcium carbonate deposits,which indicates that hic9 participates in this process and plays a key role in the formation of the macreous layer.