In order to elucidate the effect of nucleus insertion stimulation on hemolymphocytes and their function in pearl mineralization of the freshwater pearl mussel Hyriopsis cumingii, the present study explored the morphology and classification of hemolymphocytes, including four types by means of light and electron microscopy based on cellular complexity, cell size, and nucleo-cytoplasmic ratio. Giemsa staining and flow cytometry analysis showed that the granularity complexity of hemolymphocytes increased significantly, cell diameter became larger after nucleus insertion, the proportion of granulocytes increased significantly (P＜0.05), and the proportion of hyalinocyte decreased significantly (P＜0.05). The results of histological observation showed that a large number of hemolymphocytes gathered near the insertion site and granulocytes were recruited. Ca²⁺ content in hemolymphocytes and serum continued to significantly increase 2, 5, and 10 d after insertion (P＜0.05) and then significantly decreased 20 and 50 d after insertion (P＜0.05), which revealed that the number composition, distribution, and Ca²⁺ level of hemolymphocytes all respond to nucleus insertion. In vitro calcium carbonate crystallization experiments showed that hemolymphocytes and serum were able to influence the morphology of calcite but did not change the crystal shape. In summary, hemolymphocytes respond rapidly to the stimulation of nucleus insertion and can regulate biomineralization. This study lays a theoretical foundation for further research on the regulatory role of hemolymphocytes in shellfish biomineralization.