The mantle cells from Hyriopsis cumingii were obtained by using trypsin digestion. The Ca²⁺ concentrations in inner mantle cells and outer mantle cells under the resting condition and the dynamics of Ca²⁺concentration in the mantle cells incubated with various concentrations of VD₃ were measured by Fluo-3/AM fluorescent labeling and CLSM. The results showed that the average Ca²⁺ fluorescence intensity of outer mantle cells was significantly higher than that of inner mantle cells ( P <0.05). After adding different concentrations of VD_3,the average Ca²⁺ fluorescence intensity was enhanced with the increased concentration of VD₃ in culture medium, and inner mantle cells and outer mantle cells showed the same change. The difference between the control and 50 IU/L VD₃ groups, or between 500 IU/L and 1 000 IU/L groups was not significant ( P >0.05), but the difference between the control and another three groups (100 IU/L, 500 IU/L and 1 000 IU/L), or between the 50 IU/L group and another three groups(100 IU/L, 500 IU/L and 1 000 IU/L) was significant ( P <0.05). The present research may provide theoretical basis for promoting the pearl industry.