The Ca²⁺-sensing receptor (CaSR) regulates the calcium homeostasis in the human body via sensing fluctuations in the extracellular Ca²⁺ concentration. Naturally occurring mutations in the CaSR could result in Ca²⁺ regulation disorders. In the present study, we use several complementary approaches including imaging [Ca²⁺]_i response in living cells at the cellular level and using molecular dynamic (MD) simulations at the atomic level to provide important insights into the behavior of the receptor in both normal and disease statuses. We demonstrated that the molecular connectivity between [Ca²⁺]_o–binding sites is responsible for the functional positive homotropic cooperativity in the CaSR’s response to [Ca²⁺]_o. Naturallyoccurring disease mutations near Site 1 disrupted the cooperativity. We further identified an L-Phe-binding pocket adjacent to Ca²⁺-binding Site 1, which is essential for functional positive heterotropic cooperativity by having a global impact on all five of the predicted Ca²⁺-binding sites in the ECD with regards to [Ca²⁺]_o-evoked [Ca²⁺]_i signaling. Furthermore, the CaSR’s ECDs have been expressed using both bacteria and mammalian systems and were characterized using the fluorescence titration spectroscopy, circular dichroism technique as well as the NMR spectroscopy. Our studies show calcium and Phe directly bind to the ECD domain directly and interactively. Moreover, we also demonstrated that intracellular trafficking of the CaSR is a complex process, which involves modulation by calmodulin and can possibly be affected by different CaSR isoforms when expressing in various cell lines. The studies on the isolated proteins will pave the way for future protein crystallization and related structural research.