Calcitonin is a very sensitive tumor marker of medullary thyroid carcinoma (MTC). MTC patients usually have very high values of serum calcitonin that can be used to diagnose the disease. To improve the diagnostic sensitivity in family members with small MTCs and to evaluate the postoperative biochemical cure status, a calcium stimulation test is widely used. Serum calcitonin has been measured using several methods, but in Japan, only an electrochemiluminescence immunoassay (ECLIA) is currently performed to determine serum calcitonin. Reference values for the calcium stimulation test using an ECLIA have not been reported. Here we conducted a calcium stimulation tests in 26 patients without MTC before and after total thyroidectomy. Preoperatively, the basal calcitonin values of all patients were within normal limits and increased to a mean of 14.4 pg/mL after calcium stimulation. We transformed the peak values before total thyroidectomy to a logarithmic distribution and calculated the normalized mean ± 1.96× standard deviation; the reference upper limit was thus expressed. In the female patients with non-MTC, the reference upper limit was 67.6 pg/mL. In all patients, the calcium stimulation test results after total thyroidectomy showed undetectable basal and stimulated calcitonin values (<0.5 pg/mL). This is the first study to determine reference values to be used for the calcium stimulation test along with an ECLIA in non-MTC patients. We propose that female patients are regarded as biochemically cured or normal when the stimulated calcitonin values by ECLIA are <67.6 pg/mL before surgery and <0.5 pg/mL after total thyroidectomy.