Despite significant progress and notable successes in tumor therapy, malignant disease remains an extremely difficult problem in today's health care setting. There is, however, an increasing application of new therapies targeting proteins specifically upregulated on tumor cells. These innovative therapeutic approaches are aimed at molecules that contribute to malignant development and progression but spare normal tissues. The CUB domain containing protein 1 (CDCP1) is such a tumor-associated protein and, thus, a potential candidate for targeted cancer immunotherapy. Herein, we describe the generation of function-blocking human antibodies against CDCP1 that were obtained from human scFv phage display libraries using subtractive panning protocols on CDCP1 expressing cancer cells and immunopurified CDCP1 protein. One of the isolated anti-CDCP1 antibodies, namely, C20Fc, efficiently blocked experimental metastasis of human carcinoma cells, including HeLa cells stably transfected with CDCP1 and prostate carcinoma cells PC-hi/diss naturally expressing CDCP1, in both chick embryo and mouse model systems. The C20Fc antibody also reduced colony formation of CDCP1 expressing cells in a soft agar assay for anchorage-independent cell growth. Specific targeting of CDCP1 by C20Fc mediated the delivery of a toxin-conjugated antibody complex, thus, providing evidence for antibody internalization and specific killing of CDCP1-positive tumor cells. Our findings indicate a functional role for CDCP1 in human cancer and underscore the therapeutic potential of function-blocking anti-CDCP1 antibodies targeting both primary and metastatic carcinoma cells.