Overexpression of human epidermal growth factor receptor 2 (HER2) plays important roles in tumorigenesis and tumor progression in breast cancer. Nuclear imaging of HER2 expression in tumors might detect all HER2-positive tumors throughout the body and guide HER2-targeted therapies for patients. We therefore aimed to develop a HER2-targeted peptide probe for breast cancer imaging. A novel SPECT imaging probe, 99mTc-HYNIC-H6F, was prepared and then evaluated in breast cancer animal models. Methods: The HER2-targeted peptide H6F (YLFFVFER) was conjugated with the bifunctional chelator hydrazinonicotinamide (HYNIC). 99mTc-HYNIC-H6F was prepared, and the in vivo characteristics of 99mTc-HYNIC-H6F were investigated in MDA-MB-453 (HER2-positive) and MDA-MB-231 (HER2-negative) models using small-animal SPECT/CT. Moreover, to investigate the specificity of the H6F peptide toward HER2 and the potential applications in monitoring therapies involving trastuzumab, unlabeled H6F and trastuzumab were used as blocking agents in cell competition studies and SPECT imaging. Results: A standard tricine/trisodium triphenylphosphine-3,3',3″-trisulfonate labeling procedure demonstrated that the radiochemical purity was greater than 95%. 99mTc-HYNIC-H6F displayed excellent HER2-binding specificity both in vitro and in vivo. SPECT/CT imaging revealed that the MDA-MB-453 tumors were clearly visualized (percentage injected dose per gram, 3.58 ± 0.01 at 30 min after injection), whereas the signals in HER2-negative MDA-MB-231 tumors were much lower (0.73 ± 0.22 at 30 min after injection). Tumor uptake of MDA-MB-453 was blocked by the coinjection of excess H6F but not by excess trastuzumab. Conclusion: The 99mTc-HYNIC-H6F peptide probe specifically accumulates in HER2-positive tumors and is therefore promising for the diagnosis of HER2-positive cancers. Because 99mTc-HYNIC-H6F and trastuzumab target different regions of the HER2 receptor, this radiotracer also has great potential for monitoring the therapeutic efficacy of trastuzumab by rechecking the expression level of HER2 without blocking effect during therapy.