Freshwater pearls (aragonite pearls) were selected as the main experimental materials to study the biomineralization of calcium carbonate. Two kinds of soluble matrix proteins, i.e., acid soluble protein (ASM) and water soluble protein (WSM), were extracted from aragonite pearls to prepare a series of aqueous solutions with different concentrations. In vitro study was carried out by using the above_mentioned aqueous solutions with different concentrations in the diffusion method. The effects of concentrations of ASM and WSM aqueous solutions were discussed in the controlled crystallization of calcium carbonate. Scanning electron microscopy (SEM) and Raman spectroscopy were used to observe and analyze the results of in vitro biomineralization of calcium carbonate. The results show some differences in regulation and controlled crystallization of CaCO3 between ASM and WSM aqueous solutions. The concentration of ASM aqueous solutions seems to play an important role in controlling the crystal morphology and size of calcium carbonate but it has no effect on polymorphism, while the polymorph, morphology and size of crystals are obviously influenced by the concentration of WSM aqueous solutions.