Hypoparathyroidism is a rare endocrine disorder characterized by hypocalcemia, hyperphosphatemia, and low or undetectable levels of parathyroid hormone (PTH). Advanced treatments that precisely maintain blood calcium levels within the normal range to improve disease symptoms and outcomes are needed. Canvuparatide (formerly known as MBX 2109) is a once-weekly investigational PTH analog that undergoes a controlled-release conversion to a biologically active peptide through an intramolecular cyclization reaction controlled by temperature and pH. Here we demonstrate the biologically active PTH analog, derived from the canvuparatide prodrug, stimulated dose-dependent accumulation of cyclic AMP to a similar degree and selectivity as a synthetic form of the human PTH(1–34) peptide in human cells overexpressing the PTH type 1 receptor. In healthy rats treated with canvuparatide at 4–40 nmol/kg/day for 28 days and healthy cynomolgus monkeys treated with single doses of canvuparatide 3.75–7.5 nmol/kg, prodrug and active peptide concentrations increased dose proportionally and correlated with increases in serum calcium concentrations. In parathyroidectomized rats, canvuparatide treatment at 10–40 nmol/kg normalized serum calcium levels and increased bone formation in a dose-proportional manner. In a phase 1, randomized, placebo-controlled study (NCT05158335), single subcutaneously administered doses of canvuparatide (50–600 μg) were well tolerated in healthy volunteers. Pharmacokinetic clinical profiles displayed geometric mean t_1/2 values of 81–101 (canvuparatide prodrug) and 133–186 h (canvuparatide active peptide) across dose groups, supporting once-weekly dosing. These collective findings support clinical advancement of once-weekly canvuparatide therapy for patients with hypoparathyroidism.