Fetuin-A is a serum glycoprotein identified as a calcification inhibitor, and a key player in bone formation and human metabolic processes. A study on binding mechanisms of Fetuin-A with calcium carbonate nanoparticles in a simulated body fluid (DMEM) environment is presented. Observed interactions between Fetuin-A and the CaCO3 nanoparticles reveal an initial adsorption process, followed by a stabilization stage, and then a solubilization period for the Fetuin-A/CaCO3 complex. FTIR and XPS are used to monitor functional group and elemental composition changes during the initial adsorption process between Fetuin-A and the CaCO3 nanoparticles. Distinctive Fetuin-A/CaCO3 complex structures-also known as mineralo-protein particles-are imaged with TEM and SEM. DLS and UV-Vis methods are used to further characterize the in situ binding mechanisms. Results of this study can guide the design of complex organic-inorganic hybrid materials, improve current drug delivery methods, and provide insight in monitoring and controlling interactions between Fetuin-A and external calcium ions.