The negative charge of sulfotransferase can enrich Ca²⁺ and promote the nucleation and growth of calcium carbonate crystals by increasing the level of sulfation and transferring exogenous sulfonic acid groups to polysaccharide chains. Although the pearl yield of Hyriopsis cumingii is very high, the overall quality of its pearls is still different from that of seawater pearls produced abroad, which is one of the main problems that remain to be addressed in pearl culture industry. Organic macromolecules such as proteins, polysaccharides, and lipids play an important role in the process of shell biomineralization, collectively referred to as shell organic matrices. In recent years, research on the biomineralization mechanism in H. cumingii has mainly focused on the functionalities of matrix proteins, whereas the roles of polysaccharides and their respective synthesis enzymes in the biomineralization of H. cumingii remain to be elucidated. The main component of shells is calcium carbonate, which is similar to that of pearls. Thus, studying the effects of sulfotransferase on the formation of H. cumingii shells may help improve freshwater pearl quality. In this study, the sequence characteristics of the sulfotransferase gene HcCHST11 in H. cumingii were analyzed, and the potential functions of this gene in shell formation were further explored using real-time quantitative reverse transcription PCR (qRT-PCR), in situ hybridization (ISH), and RNAi and scanning electron microscopy (SEM) detection techniques. The results showed that the HcCHST11 gene open reading frame (ORF) was 840 bp long and encoded 279 amino acids, containing 1 sulfotransferase domain. The tissue expression specificity analysis found that HcCHST11 was highly expressed in the edge mantle. The in situ hybridization results showed that the positive signals were strong in the outer fold (OF), middle fold (MF), and at the connection between the middle fold and the inner fold. After interfering with HcCHST11, the expression of HcCHST11 in the edge mantle was significantly downregulated (P＜0.05). Seven days after interference, SEM detection showed that the shape of the pearl layer’s aragonite platelets became irregular, and carbonate deposits appeared on the edge. The organic sheath of the prism layer became significantly narrower, the crystal surface was rough and protruded, and cracks formed between the crystals. Functional characterization demonstrates that HcCHST11 serves as a master regulator in shell biomineralization, orchestrating the development of multilayered shell structures (periostracum, prismatic, and nacreous layers) via sulfate-dependent modulation of organic-inorganic interfaces.However, the specific mechanism by which HcCHST11 regulates shell mineralization remains unclear and needs further investigation. The precise mechanism through which the HcCHST11 gene governs shell mineralization remains undefined, and further exploration is indispensable.