<p>Mollusk shell formation is a delicate and comprehensive physiological process that relies on the precise deposition of crystals under the control of shell matrix proteins. However, the underlying protective mechanisms governing this process remain largely unknown. Herein, a novel shell matrix protein gene was identified from the freshwater mussel, <i>Hyriopsis cumingii</i>. The predicted protein is characterized by a high glycine content and two Kunitz-type SPI domains, designated as <i>Hc</i>GrKuSPI. The <i>Hc</i>GrKuSPI gene was highly expressed in the outer epithelial of the mantle edge and mantle pallial. Immunostaining in situ analysis revealed the presence of <i>Hc</i>GrKuSPI in the thick inter-prism matrix of the prismatic layer and the organic membrane of the nacreous layer. The expression of <i>Hc</i>GrKuSPI in the pearl sac significantly increased during the ordered deposition of pearl nacre tablets. The resultant recombinant protein, SUMO-<i>Hc</i>GrKuSPI, exhibited a strong affinity for aragonite and calcite and was involved in the morphological modification of calcite crystals in vitro. These results indicate that <i>Hc</i>GrKuSPI is involved in organic framework construction and crystal morphological modification during the prismatic and nacreous layers formation. Furthermore, the expression of <i>Hc</i>GrKuSPI in the mantle significantly increased following bacterial infection within the extrapallial fluid, and SUMO-<i>Hc</i>GrKuSPI exhibited a strong inhibitory effect against bacterial growth and trypsin activity. Antibody injection in vivo led to severe damage to the thick inter-column framework of the prismatic layer and morphological deformities of nacre tablets. These findings indicate that <i>Hc</i>GrKuSPI exhibits excellent protease inhibitory and antibacterial activities, providing a matrix protection system to ensure the smoothness of shell and pearl formation. Overall, these results enhance our understanding of the protective mechanisms involved in mollusk biomineralization.</p>