Metastasis, which remains incompletely characterized at the molecular and biochemical levels, is a highly specific process. Despite the ability of disseminated cancer cells to intravasate into distant tissues, it has been long recognized that only a limited subset of target organs develop clinically overt metastases. Therefore, subsequent adaptation of disseminated cancer cells to foreign tissue microenvironment determines the metastatic latency and tissue tropism of these cells. As a result, studying interactions between the disseminated cancer cells and the adjacent stromal cells will provide a better understanding of what constitutes a favorable or unfavorable microenvironment for disseminated cancer cells in a tissue-specific manner. Previously, we reported a protein signature of brain metastasis showing increased ability of brain metastatic breast cancer cells to counteract oxidative stress. In this study, we showed that another protein from the brain metastatic protein signature, neurotrophin-3 (NT-3), has a dual function of regulating the metastatic growth of metastatic breast cancer cells and reducing the activation of immune response in the brain. More importantly, increased NT-3 secretion in metastatic breast cancer cells results in a reversion of mesenchymal-like (EMT) state to epithelial-like (MET) state and vice versa. Ectopic expression of NT-3 in EMT-like breast cancer cells reduces their migratory ability and increases the expression of HER2 (human epidermal growth factor receptor 2) and E-cadherin at the cell–cell junction. In addition, both endogenous and ectopic expression of NT-3 reduced the number of fully activated cytotoxic microglia. In summary, NT-3 appears to promote growth of metastatic breast cancer cells in the brain by facilitating the re-epithelialization of metastatic breast cancer cells and downmodulating the cytotoxic response of microglia. Most importantly, our results provide new insights into the latency and development of central nervous system macrometastases in patients with HER2-positive breast tumors and provide mechanistic rationale to target HER2 signaling for HER2-positive breast cancer brain metastasis.