Abstract: Calcium concentration has crucial effects on the growth and nacre deposition of the freshwater pearl mussel Hyriopsis cumingii. However, the suitable Ca2+ concentration for the mussel culture and nacre deposition has been controversial. In this paper, we investigated how the Ca2+ concentration regulated the growth of Hyriopsis cumingii, the nacre deposition, and the nacre crystal structure. Pearl mussels were co-cultured with fish under different Ca2+ concentrations from 10 to 80 mg/L. The results showed that 1-year-old mussels grew fastest under the Ca2+ concentration of 40 mg/L. The suitable Ca2+ concentration range for the growth of 2-year-old non-implanted mussels were 40 mg/L to 70 mg/L. The suitable Ca2+ concentration for the pearl deposition of 2-year-old implanted mussels was 40 mg/L. Using Raman spectra and scanning electron microscope we showed that Ca2+ at the suitable concentration facilitated the nacre deposition of pearl mussels through promoting the secretion of organic matrix and the crystallization and growth of CaCO3. Our results implied that adding exogenous calcium, such as quicklime, into the water could improve the growth of the mussels and pearls. This method is potentially a new tool to accelerate the pearl cultivation and to improve the pearl quality.