Biomineralization occurs in aqueous environments. Despite the ubiquity and relevance of CaCO biomineralization, the role of water in the biomineralization process has remained elusive. Here, we demonstrate that water reorganization accompanies CaCO biomineralization for sea urchin spine generation in a model system. Using surface-specific vibrational spectroscopy, we probe the water at the interface of the spine-associated protein during CaCO mineralization. Our results show that, while the protein structure remains unchanged, the structure of interfacial water is perturbed differently in the presence of both Ca and CO compared to the addition of only Ca. This difference is attributed to the condensation of prenucleation mineral species. Our findings are consistent with a nonclassical mineralization pathway for sea urchin spine generation and highlight the importance of protein hydration in biomineralization.