As a natural calcium resource, animal bone needs to be miniaturized to the nanoscale to improve palatability and absorption capacity. To explore the mechanism of high-pressure homogenization (HPH) in preparing natural bone aqueous nanosuspensions, the relationships between the changes in protein conformation, solubility and quality characteristics of rabbit bone aqueous suspensions (RBAS) prepared by different HPH cycles were studied. The results showed that the improvements in particle size, stability and calcium solubility of RBASs could be mainly attributed to the improvement of protein solubility induced by the changes in protein conformation. HPH treatment led to the denaturation and degradation of protein in rabbit bone, generating soluble peptides and improving the stability of the suspensions by enhancing the surface charge of the particles. When collagen as the main protein was partially degraded, the hydroxyapatite in the bone was crushed into tiny particles. The increase in the particle-specific surface area led to the release of calcium ions, which chelated with the peptides to produce peptide calcium. However, excessive HPH treatment caused the production of protein macromolecular aggregates and affected the quality of RBASs. This study is helpful to promote the application of HPH technology in animal bone nanoprocessing.