Background and Objectives. Sentinel lymph node biopsy has been widely accepted as the standard procedure to assess the axillary lymph node status in breast cancer. However, more than 70% of excised sentinel lymph nodes have been found to be healthy. It may lead to unnecessary excisions and increase the incidence of postoperative complications. The aim of this study was to investigate the value of quantitative Tc-99 m sulfur colloid SPECT/CT in avoiding excessive removal of unnecessary sentinel lymph nodes in breast cancer patients. Methods. We retrospectively enrolled breast cancer patients who underwent SPECT/CT prior to sentinel lymph node biopsy. Quantification of radiotracer uptake from SPECT/CT data was performed. A radioactivity count threshold (RSPECT) using SPECT/CT was calculated for detecting metastatic sentinel lymph nodes. To localize sentinel lymph nodes exactly, we compared the positions of sentinel lymph nodes localized using SPECT/CT with positions localized surgically using an intraoperative γ-probe. Results. 491 patients were included, with a median of 3 sentinel lymph nodes/patient detected by the γ-probe and 2 sentinel lymph nodes/patient detected by SPECT/CT. As the number of sentinel lymph nodes visualized on SPECT/CT images, the metastasis incidence of lymph nodes in the ≤2 SLNs group was significantly higher than that in the >2 SLNs group (35% vs. 15%,
P
<
0.001
). No metastasis was found in lymph nodes with RSPECT ≤ 30% in the >2 SLNs group, and thus, 30% (157/526) of SPECT/CT-identified nodes would avoid unnecessary removal. The positions of sentinel lymph nodes localized by SPECT/CT and γ-probe were identical in 42% (39/93) of patients. Conclusions. Quantitative Tc-99 m SC SPECT/CT imaging has the potential to preoperatively locate sentinel lymph nodes and intraoperatively avoid unnecessary sentinel lymph node biopsy.