Human extracellular matrix protein-1 (hECM1), a secreted glycoprotein, is widely expressed in different tissues and organs. ECM1 has been implicated in multiple biological functions, which are potentially mediated by the interaction of different ECM1 domains with its ligands. However, the exact biological functions of ECM1 have not been elucidated yet, and the functional study of ECM1 has been partially hampered by the lack of sensitive and specific antibodies, especially those targeting different ECM1 domains. In this study, six strains of monoclonal antibody (MAb) against hECM1 were generated using purified, prokaryotically-expressed hECM1 as an immunogen. The MAbs were shown to be highly sensitive and specific, and suitable for western blot, immunoprecipitation assays and immunohistochemistry. Furthermore, the particular ECM1 domains recognized by different MAbs were identified. Lastly, the MAbs were found to have neutralizing activities, inhibiting the proliferation, migration and metastasis of MDA-MB-231 cells. In conclusion, the domain-specific anti-ECM1 MAbs produced in this study should provide a useful tool for investigating ECM1's biological functions, and cellular pathways in which it is involved. (C) 2016 Elsevier Inc. All rights reserved.