Homotypic aggregation (HA) of cells plays key roles in physiological and pathological processes, such as embryogenesis, immune responses, angiogenesis, tumor cell invasion, and metastasis. Aminopeptidase N (CD13) has been implicated in most of these phenomena, although its participation has been attributed to its enzymatic activity, while its role as an adhesion molecule has been almost unexplored. Here, we show that certain anti-CD13 monoclonal antibodies induce HA of monocytic U-937 cells, independently of their effect on enzymatic activity. The phenomenon is related to binding to a specific site on the CD13 molecule and is independent of integrins. It is abrogated by low temperature, by the glycolysis inhibitor 2-deoxyglucose, and by inhibitors of tyrosine and mitogen-activated protein kinases. The inhibitor of microtubule polymerization colchicine has a synergistic effect on CD13-mediated aggregation, suggesting an inhibitory role of microtubules in this process. Finally, during HA, CD13 actively redistributes to the zones of cell-cell contact, as determined by live cell imaging studies, demonstrating a direct role of CD13 in the adhesion phenomenon. Together, these data show for the first time the participation of CD13 in monocytic cell adhesion.