Background: To investigate the role of parathyroid technetium-99m-hexakis2-methoxy-2-methylpropylisonitrile (99mTc-MIBI) single photon emission computed tomography/ computed tomography (SPECT/CT) combined with the serum calcium (Ca) and serum parathyroid hormone (PTH) in the differential diagnosis of MIBI uptake lesion. Methods: 201 patients with MIBI uptake lesion on parathyroid 99mTc-MIBI SPECT/CT from January 2015 to July 2019 were enrolled in this study. All patients who underwent surgical resection were classified into two groups: primary hyperparathyroidism (PHPT) and non-PHPT in terms of the pathological findings. Radiological performance of 99mTc-MIBI SPECT/CT, serum Ca and serum PTH were comparable between the two groups.Results: 201 patients (135 females; median age, 53.0 years; age range, 29 – 79 years) were included. Pathological findings were as follows: PHPT was in 126 (62.7%) patients, including parathyroid adenoma in 106 patients, parathyroid cancer in 12 patients and parathyroid hyperplasia in 8 patients, while non-PHPT were in 75 (37.3%) patients, including thyroid adenoma in 14 patients, thyroid papillary cancer in 15 patients and thyroid nodular goiter in 46 patients. In the following multivariable logistic regression analysis, serum Ca and diameter of the shortest axis of the lesion were the independent factors for differentiating PHPT from non-PHPT. In receiver operating characteristic (ROC) analyses, the cut-off value of serum Ca differentiating PHPT from non-PHPT was 2.6 mmol/L, yielding the area under the ROC curve (AUC) of 0.931, sensitivity of 85.7%, specificity of 89.2%; the cut-off value of diameter of the shortest axis of the lesion was 20.4mm, yielding AUC of 0.728, sensitivity of 62.2%, specificity of 87.1%.Conclusion: Parathyroid 99mTc-MIBI SPECT/CT combined with serum Ca and serum PTH contributed to the differential diagnosis of PHPT from non-PHPT, evenly can assist the determination of the specific pathology of MIBI uptake lesion before surgery.