The Calcium-Sensing Receptor (CaSR) integrates diverse nutrient signals, including Ca²⁺ and amino acids, to regulate systemic calcium homeostasis. Dysfunction of CaSR leads to familial hypocalciuric hypercalcemia type 1, neonatal severe hyperparathyroidism, and autosomal dominant hypocalcemia. Currently, four positive allosteric modulators (PAMs) targeting CaSR have been approved for treating these disorders; however, they often cause side effects such as calciuria and renal complications. Additionally, CaSR harbors over 600 disease‑associated genetic variants, with at least 292 inactivating missense mutations, which limit the clinical efficacy of existing PAMs and highlight the pressing need for next‑generation therapeutics. To address this, we developed a FRET‑based conformational biosensor to elucidate the key conformational changes underlying CaSR activation. We discovered that Ca²⁺ alone is sufficient to fully stabilize the active conformation, whereas L‑amino acids function exclusively as pure PAMs. Furthermore, we demonstrated that inter‑subunit disulfide bridges are essential for maintaining the inactive state, as their absence results in undetectable constitutive activity; notably, human mutations deleting these bridges, which are associated with hypocalcemia, elevate constitutive activity. Finally, we generated two nanobodies that act as PAMs by binding to a novel allosteric pocket. These nanobodies effectively suppress parathyroid hormone secretion in primary human parathyroid cells, act additively with cinacalcet, and restore Ca²⁺ sensitivity across a range of disease‑associated loss‑of‑function variants. Collectively, our findings provide a novel strategy for CaSR modulation and support a promising nanobody‑based therapeutic approach for patients carrying receptor genetic variants