Plague is an acute infection caused by the Gram-negative bacterium Yersinia pestis. Antibodies that are protective against plague target LcrV, an essential virulence protein and component of a type III secretion system of Y. pestis. Secreted LcrV localizes to the tips of type III needles on the bacterial surface, and its function is necessary for the translocation of Yersinia outer proteins (Yops) into the cytosol of host cells infected by Y. pestis. Translocated Yops counteract macrophage functions, for example, by inhibiting phagocytosis (YopE) or inducing cytotoxicity (YopJ). Although LcrV is the best-characterized protective antigen of Y. pestis, the mechanism of protection by anti-LcrV antibodies is not fully understood. Antibodies bind to LcrV at needle tips, neutralize Yop translocation, and promote opsonophagocytosis of Y. pestis by macrophages in vitro. However, it is not clear if anti-LcrV antibodies neutralize Yop translocation directly or if they do so indirectly, by promoting opsonophagocytosis. To determine if the protective IgG1 monoclonal antibody (MAb) 7.3 is directly neutralizing, an IgG2a subclass variant, a deglycosylated variant, F(ab')2, and Fab were tested for the ability to inhibit the translocation of Yops into Y. pestis-infected macrophages in vitro. Macrophage cytotoxicity and cellular fractionation assays show that the Fc of MAb 7.3 is not required for the neutralization of YopJ or YopE translocation. In addition, the use of Fc receptor-deficient macrophages, and the use of cytochalasin D to inhibit actin polymerization, confirmed that opsonophagocytosis is not required for MAb 7.3 to neutralize translocation. These data indicate that the binding of the variable region of MAb 7.3 to LcrV is sufficient to directly neutralize Yop translocation.