We report the application of quantitative mass spectrometry to identify plasma membrane proteins differentially expressed in melanoma cells with high vs. low metastatic abilities. Using stable isotope labeling with amino acids in culture (SILAC) coupled with nanospray tandem mass spectrometry, we identified CUB-domain-containing protein 1 (CDCP1) as one such differentially expressed transmembrane protein. CDCP1 is not only a surface marker for cells with higher metastatic potential, but also functionally involved in enhancing tumor metastasis. Overexpression of CDCP1 also correlates with activation of Src. Pharmacological reagents, PP2 and Dasatinib, which block Src family kinase activation, blocked scattered growth of CDCP1-overexpressing cells in 3D Matrigel culture, suggesting that CDCP1 might function through the activation of Src-family kinases (SFKs). This hypothesis was further supported by mutational studies of CDCP1. Whereas wild-type CDCP1 enhances Src activation, point mutation Y734F abolishes in vitro dispersive growth in 3D culture and in vivo metastasis-enhancing activities of CDCP1. In addition, the Y734F mutation also eliminated enhanced Src activation. Thus, this work provides molecular mechanisms for the metastasis-enhancing functions of CDCP1.