The vascular cell adhesion molecule-1 (VCAM-1) plays an important role in inflammation, where it facilitates the recruitment of leukocytes to the inflamed area via leukocytes’ VLA-4 and endothelial cells’ VCAM-1 interaction. VCAM-1 expression is also upregulated in certain cancers. VCAM-1 has seven Ig-like domains, with domains 1 and 4 shown to be critical for VLA-4 binding. However, the specific functions of individual VCAM-1 Ig-like domains remain poorly understood. In this study, we identified single-chain variable fragment (scFv) antibodies targeting domains 2, 3, and 5 of VCAM-1, and investigated the ability of these antibodies to block VCAM-1-mediated cell adhesion to macrophages. We show that scFv antibodies against Ig-like domains 2 and 3 interfere with the ability of macrophages to bind endothelial cells, suggesting that these domains also play a role in facilitating this interaction. These results emphasize the need to more carefully study the role of each domain on VCAM-1 function and highlight the potential of targeting these VCAM-1 domains for more tailored therapeutic interventions in inflammatory diseases and cancer.