Biomineralization is the study of minerals produced by biomaterials or living organisms, and it has attracted considerable attention in industry and fundamental research. Here, for the first time, the roles of alkali-treated mung bean (MB) and cassava (CS) starches as templates for calcium carbonate (CaCO3) spherulite formation were investigated by bulk rheology and molecular dynamics (MD) simulations. The rheological results, including yield stress, flow behavior index, consistency coefficient, and hysteresis area of alkali-treated MB and CS starch suspensions containing Ca2+, revealed that the Ca2+ induced the folding of starch chains via Ca2+ crosslinking, leading to aggregation of starch-Ca2+ complexes. After the starch films were fabricated from the suspensions, CaCO3 spherulites were observed inside the films. The size and number of the spherulites increased with increasing concentration of Ca2+ in the suspensions as a result of an increase in nucleation sites needed for spherulite growth. Moreover, to gain a deeper understanding of the interactions between Ca2+ and starch and of the formation of the nucleation sites for spherulite growth, MD simulations were performed. Our findings pave the way for the design of new biomaterial-based templates, which can efficiently control crystal shape and size of CaCO3.