Integrin α6 associates with the survival and migration of breast cancer cells, which emerges as a predictor of reduced overall survival and worse prognosis. In this study, we developed an integrin α6-targeted radiotracer 99mTc-RWY for SPECT imaging of breast cancer. The safety, biodistribution and radiation dosimetry were studied in seven healthy volunteers, and the clinical potential of 99mTc-RWY was examined in two breast cancer patients. A single dose of 11.1 MBq/kg 99mTc-RWY was injected intravenously. Visual and semiquantitative methods were used to assess the SPECT/CT images. The routine Hematoxylin-Eosin (HE) staining and immunohistochemistry (IHC) staining were subsequently carried out to confirm the Integrin α6 expression of tumors.