Antibody-dependent cell cytotoxicity (ADCC) plays a critical role in monoclonal antibody (mAb)-mediated cancer therapy. ADCC, however, has not been directly shown in vivo but inferred from the requirement for IgG Fc receptors (FcγR) in tumor rejection in mice. Here, we investigated the mechanism of action of a Tn antigen-specific chimeric mAb (Chi-Tn), which binds selectively to a wide variety of carcinomas, but not to normal tissues, in both humans and mice. Chi-Tn mAb showed no direct toxicity against carcinomas cell lines in vitro but induced the rejection of a murine breast tumor in 80% to 100% of immunocompetent mice, when associated with cyclophosphamide. Tumor rejection was abolished in Fc receptors-associated γ chain (FcR-γ)-deficient mice, suggesting a role for ADCC. Indeed, tumor cells formed stable conjugates in vivo with FcR-γ chain-expressing macrophages and neutrophils in Chi-Tn mAb-treated but not in control mAb-treated mice. The contact zone between tumor cells and ADCC effectors accumulated actin, FcγR and phospho-tyrosines. The in vivo formed ADCC synapses were organized in multifocal supra-molecular activation clusters. These results show that in vivo ADCC mediated by macrophages and neutrophils during tumor rejection by Chi-Tn mAb involves a novel type of multifocal immune synapse between effectors of innate immunity and tumor cells.