Brain metastases are a common feature of malignant melanoma and are associated with poor prognosis. Melanotransferrin (MTf), one of several antigens associated with the surface of melanoma cells, has been demonstrated to promote cell invasion. In this study, we investigated the role of membrane-bound MTf in several of the steps leading to the development of melanoma brain metastasis. Our results indicated that MTf-positive cells were detected in the brains of nude mice injected intravenously with human melanoma SK-Mel 28 cells. Moreover, administration of a single dose of a monoclonal antibody (L235) directed against human MTf significantly reduced the development of human melanoma brain metastases in nude mice. The ability of melanoma cells to cross the blood-brain barrier (BBB) in vitro is correlated with their MTf expression levels at the cell surface. Overall, our results indicated that membrane-bound MTf is a key element in melanoma cell transmigration across the BBB and subsequent brain metastasis. Thus, these data suggest MTf as an attractive target and demonstrate the therapeutic potential of an anti-MTf mAb for preventing metastatic melanoma.