The quality of pearls is related closely to the position where the nucleus is inserted.The visceral mass of freshwater pearl mussels has the physiological condition for the culture of pearls, but the appropriate position for inserting the nucleus has not been determined. In this study, the physiological responses and cultured pearl biology were used to explore the most suitable of five positions in the visceral mass for inserting the nuclei (I: the front end axe foot visceral mass, II: the central axe foot visceral group, III: near the gonad department, IV: near the stomach and V: near the kidney).Overall, kidney damage (urea nitrogen/BUN, urine creatinine/Cr, uric acid/UA) and calcium metabolism indicators were in decline, whereas liver damage indicators (aspartate aminotransferase/AST, alanine aminotransferase/ALT) rose. Enzyme activity index was determined by enzyme-linked immunosorbent assay. These results indicated that the insertion caused metabolic disorders and immune inflammation. The axe foot visceral group was not significant that compared with the control group, so it was suitable for nucleation. The peak of granulocytes and hyalinocytes in the blood cells appeared on day 10 (thd) and exerted an early immune defence function.Furthermore, the granulosa synthesized by the epidermal cells of the pearl capsule was in the mature stage of normal nacre secretion, thus forming a thicker deposit of pearls. This study filled the gaps in knowledge of visceral mass cultivating pearls and provided a theoretical basis for artificial pearl breeding.