Despite high vaccination rates, the incidence of whooping cough has been steadily increasing in developing countries for several decades. The current acellular pertussis (aP) vaccines all include the major protective antigen pertussis toxin (PTx) and are safer, but appear less protective than infection or older whole cell vaccines. To better understand the attributes of individual antibodies stimulated by aP, we isolated plasmablast clones recognizing PTx after booster immunization of two donors. Five unique antibody sequences recognizing native PTx were recovered and expressed as recombinant human IgG1 antibodies. The antibodies all bind different epitopes on the PTx S1-subunit, B-oligomer or the S1-B subunit interface and just one clone neutralized PTx in anin vitroassay. To better understand the epitopes bound by the non-neutralizing S1-subunit antibodies, comprehensive mutagenesis with yeast display provided a detailed map of the epitope recognized by antibodies A8 and E12. Residue R76 is required for antibody A8 binding and is present on the S1 surface but is only partially exposed in the holotoxin, providing a structural explanation for A8’s inability to neutralize holotoxin. The B-subunit specific antibody D8 inhibited PTx binding to a model receptor and neutralized PTxin vitroas well as anin vivoleukocytosis assay. This is the first study to identify individual human antibodies stimulated by the acellular pertussis vaccine and demonstrates the feasibility of these approaches to address outstanding issues in pertussis vaccinology, including mechanisms of accelerated waning protective immunity despite repeated aP immunization. Copyright 2018 American Society for Microbiology.