CCN1, a secreted matrix-associated molecule, is involved in multiple cellular processes. Accumulating evidence supports that CCN1 plays an important role in tumorigenesis and progression of breast cancer. In this study, we have developed a novel CCN1 function-blocking monoclonal antibody (mAb), designated YM1B. YM1B binds to human CCN1 with high specificity, recognizing the native CCN1 structure with undisturbed disulfide linkages. Our analyses have mapped the YM1B recognition region to domain IV of CCN1, likely in proximity to the DM site. In breast cancer cells, CCN1 can induce actin reorganization, formation of lamellipodia, and cell migration/invasion through the alpha V integrins/Rac1/ERK signaling axis; these CCN1-dependent activities can be effectively suppressed by YM1B. Our results also suggest that YM1B may exert its CCN1-blocking effect by perturbing the interaction of CCN1 with vitronectin and fibronectin, which are ligands of alpha V integrins and instrumental for integrin activation. This CCN1-specific mAb may open a new potential avenue for therapeutic intervention of breast cancer progression. (C) 2013 Elsevier Inc. All rights reserved.