Objective To validate the quantitative accuracy of 99Tcm-pertechnetate(99TcmO4-)SPECT/CT imaging and its influencing factors (sphere size, radioactive concentration, reconstruction methods). Methods National Electrical Manufacturers Association (NEMA) International Electrotechnical Commission (IEC) body phantom with 99TcmO4- solution filled in 6 spheres (diameter: 10, 13, 17, 22, 28, 37 mm) and 1 cylinder (target/background ratio: 10∶1) was used to assess the accuracy of uptake quantification. SPECT/CT was used for imaging data acquisition, and volume of interest (VOI) was drawn after the images being corrected and reconstructed. The radioactive concentrations of 6 spheres were measured and compared with the true radioactive concentrations, and quantitative accuracy of each sphere was calculated. The experiment was repeated for 12 times with the same method and different radioactive concentrations. Ordered-subsets expectation maximization (OSEM) reconstruction was performed with 2 or 5 iterations to process imaging data. CT VOI and nuclear medicine (NM) threshold methods were used for the delineation of VOI. Accuracy was compared by paired t test. Results The NM thresholds to get calculated volume close to the true volume were determined to be 40%, 40%, 50%, 70%, 80%, 90% for 6 spheres with diameters of 37, 28, 22, 17, 13, 10 mm, respectively. Comparing 2 methods of VOI delineation (CT VOI and NM threshold), only the accuracy for 17 mm sphere was significantly different (t=5.89, P<0.01). Five iterations group showed higher accuracy than 2 iterations group (t values: 3.19-6.92, all P<0.05). The average accuracies under 12 different radioactive concentrations were 102%, 95%, 80%, 66%, 27%, 20%, respectively, for spheres in order from large size to small size. Conclusion The sphere volume and reconstruction method have effect on quantitative accuracy of 99TcmO4- SPECT/CT imaging, and spheres smaller than 17 mm is also significantly affected by partial volume effect. Key words: Tomography, emission-computed, single-photon; Tomography, X-ray computed; Models, structural; Image processing, computer-assisted; Sodium pertechnetate Tc 99m