Brain metastatic breast cancer (BMBC) is uniformly fatal and increasing in frequency. Despite its devastating outcome, mechanisms causing BMBC remain largely unknown. The mechanisms that implicate circulating tumor cells (CTCs) in metastatic disease, notably in BMBC, remain elusive. We characterize CTCs isolated from peripheral blood mononuclear cells of patients with breast cancer and also develop CTC lines from three of these patients. In epithelial cell adhesion molecule (EpCAM)-negative CTCs, we identified a potential signature of brain metastasis comprising "brain metastasis selected markers (BMSMs)" HER2+ / EGFR+ / HPSE+ / Notch1+. These CTCs, which are not captured by the CellSearch platform because of their EpCAM negativity, were analyzed for cell invasiveness and metastatic competency in vivo. CTC lines expressing the BMSM signature were highly invasive and capable of generating brain and lung metastases when xenografted in nude mice. Notably, increased brain metastatic capabilities, frequency, and quantitation were detected in EpCAM- CTCs overexpressing the BMSM signature. The presence of proteins of the BMSM CTC signature was also detected in the metastatic lesions of animals. Collectively, we provide evidence of isolation, characterization, and long-term culture of human breast cancer CTCs, leading to the description of a BMSM protein signature that is suggestive of CTC metastatic competency to the brain.