Living organisms can produce elegant structures with unique functions and properties through biological processes. Various proteins are involved in these processes. Inspired by the structure formation of mollusc shells, a single multifunctional recombinant protein ChiCaSifi was designed on the basis of mineralization proteins for regulating CaCO3 mineralization in a simple and direct manner. ChiCaSifi contains functional domains of the chitin binding protein (Chi), the calcium binding protein (Ca), and the silk fibroin (Sifi). Therefore, ChiCaSifi can have multiple roles in directing CaCO3 mineralization. Overexpression and purification of ChiCaSifi were achieved. Activities of ChiCaSifi were examined for its binding to calcium and chitin. Influences of ChiCaSifi in regulating the phase formation of CaCO3 crystals on the chitin surface were proved. Structural changes of ChiCaSifi were evidenced and related to its functions on mineralization. These observations indicate that rationally designed proteins with functional domains of mineralization proteins can be effective tools in materials synthesis. The present study may not only provide an insight into the formation of natural biomaterials, but also open a new avenue in the design and synthesis of novel organic–inorganic composite materials.