Significance Blood Ca ²⁺ homeostasis is maintained by the actions of the parathyroid hormone (PTH) on its cognate receptor PTHR. PTH binding to PTHR mediates prolonged adenosine 3′,5′-cyclic monophosphate (cAMP) responses in cells after receptor internalization. Here, we show that extracellular Ca ²⁺ prolongs the residence time of ligands on the receptor, consequently, increasing both the duration of receptor activation and cAMP signaling. This positive Ca ²⁺ allostery is lost for the PTH mutant R25C, recently identified as a new cause of hypocalcemia in humans. Using mass spectrometry approaches, we identify acidic clusters within the first extracellular loop of PTHR as determinants of Ca ²⁺ allostery and endosomal cAMP signaling. These findings provide insight into the molecular etiology of hypocalcemia and disease relevance of endosomal cAMP signaling.