Colorectal cancer (CRC) is a major health problem. Biomarkers associated with molecular changes in cancer cells can aid early detection, diagnosis, prognosis, therapy selection, and disease monitoring. Tumor tissue secretomes are a rich source of candidate biomarkers. To identify CRC protein biomarkers, secretomes of four pairs of human CRC tissue and patient-matched normal colon tissue samples, and secretomes of five CRC cell lines were analyzed by GeLC-MS/MS. Subsequent data analysis was based on label-free spectral counting, Ingenuity Pathway Analysis, Secretome/SignalP, STRING and Cytoscape, resulting in 2703 protein identifications in the tissue secretomes, of which 409 proteins were significantly more present in CRC samples than in controls. Biomarker selection of 76 candidates was based on consistent and abundant over-representation in cancer- compared to control-secretomes, and presumed neoplastic origin. Overlap analysis with previously obtained datasets revealed 21 biomarkers suited for early detection of CRC. Immunohistochemistry confirmed overexpression in CRC of one candidate marker (MCM5). In conclusion, a human reference dataset of 76 candidate biomarkers was identified for which we illustrate that combination with existing pre-clinical datasets allows pre-selection of biomarkers for blood- or stool-based assays to support clinical management of CRC. Further dedicated validation studies are required to demonstrate their clinical applicability. Tissue secretome proteomes are a rich source of candidate biomarkers. Several secretome proteome datasets have been obtained from pre-clinical in vitro and in vivo colorectal cancer (CRC) model systems, yielding promising CRC biomarkers obtained under well-defined experimentally controlled conditions. However, which of these biomarker proteins are actually secreted by human CRC samples was not known. To our knowledge, this is the first study that directly compares secretome proteomes from clinically relevant human CRC tissues to patient-matched normal colon tissues. We identified 76 human CRC protein biomarkers that may facilitate blood-based or stool-based assay development to support clinical management of CRC. Overlap analysis with datasets from well-defined pre-clinical studies helps to determine what clinical application suits these human CRC biomarkers best, i.e. early detection, diagnosis, prognosis, therapy selection, and/or disease monitoring of CRC. This is demonstrated for a CRC mouse model dataset, revealing 21 human CRC biomarkers suited for early detection of CRC. Copyright © 2014 Elsevier B.V. All rights reserved.