Significance Two major serotypes of Vibrio cholerae circulate globally and cause pandemic cholera, but how infection or vaccination with each serotype impacts immune responses and subsequent protection against future infection by the pathogen is not well-known. We engineered matched serotype variants of a new oral cholera vaccine candidate, HaitiV, to investigate the effect of serotype variation on vaccine immunogenicity and protective efficacy. Our results provide a rigorous framework for oral bacterial vaccine evaluation in vivo and illuminate how serotype influences oral cholera vaccine potency, a finding of significant translational interest.