Objective Medullary thyroid carcinoma (MTC) can be very aggressive, and early diagnosis is based on routine measurement of serum calcitonin (CT) and RET genetic testing for hereditary forms. Basal serum CT (bCT) concentrations are useful in the early detection of MTC, although it is still unclear whether they can also be used for the differential diagnosis between MTC and C-cell hyperplasia (CCH). Since false-positive results can be obtained with the basal measurement of CT, a provocative test to evaluate stimulated CT (sCT) is often needed. The objective of this study was to investigate the utility of a calcium gluconate test for CT in distinguishing MTC from CCH, a precancerous condition in hereditary forms of MTCs but with unclear significance in sporadic MTCs. Methods A total of 74 patients underwent the calcium loading test before thyroidectomy, and bCT and sCT levels were compared with histologic results by receiver operating characteristic plot analyses. Results A peak CT level of 388.4 pg/mL after stimulation with calcium gluconate was able to significantly distinguish patients with MTC from those with CCH and those without C-cell pathology, with 81.8% sensitivity and 36.5% specificity. A bCT level of 16.1 pg/mL was able to distinguish between these 2 groups of patients with a sensitivity of 90%. Conclusion High-dose calcium test is an effective procedure that can be applied for differential diagnosis of MTC and CCH. Reference ranges for calcium sCT levels and CT thresholds in different groups of patients have been identified.