Pertussis toxin (PTx) is a major protective antigen produced by Bordetella pertussis that is included in all current acellular vaccines. Of several well-characterized monoclonal antibodies binding this toxin, the humanized hu1B7 and hu11E6 antibodies are highly protective in multiple in vitro and in vivo assays. In this study, we determine the molecular mechanisms of protection mediated by these antibodies. Neither antibody directly binds the B. pertussis bacterium nor supports antibody-dependent complement cytotoxicity. Both antibodies, either individually or as a cocktail, form multivalent complexes with soluble PTx that bind the FcRIIb receptor more tightly than antibody alone, suggesting the antibodies may accelerate PTx clearance via immune complex formation. However, a receptor binding assay and cellular imaging indicate that the main mechanism used by hu11E6 is competitive inhibition of PTx binding to its cellular receptor. In contrast, the main hu1B7 neutralizing mechanism appears to be inhibition of PTx internalization and retrograde trafficking. We assessed the effects of hu1B7 on PTx retrograde trafficking in CHO-K1 cells using quantitative immunofluorescence microscopy. In the absence of hu1B7 or after incubation with an isotype control antibody, PTx co-localizes to organelles in a manner consistent with retrograde transport. However, after pre-incubation with hu1B7, PTx appears restricted to the membrane surface with co-localization to organelles associated with retrograde transport significantly reduced. Together, these data support a model whereby hu11E6 and hu1B7 interfere with PTx receptor binding and PTx retrograde trafficking, respectively. This article is protected by copyright. All rights reserved.