Background: This study sought to establish a rat model of hypoparathyroidism by removing the rat parathyroid glands, and compare the effects of different transplantation sites and transplantation methods using a primary culture of parathyroid cells in vitro on the hormone secretion of the model rats. Methods: Male Sprague Dawley (SD) rats were selected for in vivo parathyroid gland removal, and rats with abnormal postoperative water intake, weight gain, parathyroid hormone (PTH), and blood calcium ion concentration were selected as transplant recipients and divided into the model group, brachioradialis muscle cell transplantation group, gelatin sponge group, and subcutaneous transplantation group. The parathyroid tissue was removed and the primary cell culture was performed in vitro using homozygous SD rats as graft donors. When the parathyroid cells were able to secrete PTH, transplantation was performed, and the postoperative recovery of the PTH function of the rats with different transplantation sites and methods were observed. Results: A recipient model with low PTH was successfully established, and parathyroid progenitor cells with obvious PTH secretion were obtained. Better secretion was observed in the brachioradialis cell group compare with other groups. Conclusions: The in vitro primary cell culture of the donor parathyroid cells combined with cell transplantation significantly improved the physiological function of the hypoparathyroid rats, and could potentially replace traditional clinical brachioradialis muscle tissue transplantation.