Recent efforts to develop an enterotoxigenic Escherichia coli (ETEC) vaccine have focused on the antigenically conserved tip adhesins of colonization factors. We showed previously that intranasal immunization with dsc19CfaE, a soluble variant of the in cis donor strand complemented tip adhesin of CFA/I fimbria, is highly immunogenic and protects against oral challenge with CFA/I+ ETEC strain H10407 in the Aotus nancymaae non-human primate. We also reported a cholera toxin (CT)-like chimera (called dsc19CfaE-CTA2/CTB) in which the CTA1 domain of CT was replaced by dsc19CfaE that was strongly immunogenic when administered intranasally or orogastrically in mice. Here we evaluate the immunogenicity and protective efficacy of a refined and more stable chimera comprised of a pentameric B-subunit of ETEC heat-labile toxin (LTB) in lieu of the CTB pentamer and a donor strand truncation (dsc14) of CfaE. The refined chimera, dsc14CfaE-sCTA2/LTB, was highly immunogenic in mice when administered intranasally or intradermally, eliciting serum and fecal antibody responses against CfaE and LTB, as well as strong hemagglutination inhibition titers, a surrogate for neutralization of intestinal adhesion mediated by CfaE. Moreover, the chimera was safe and highly immunogenic when administered intradermally to guinea pigs. In A. nancymaae, ID immunization with chimera + LT(R192G) elicited strong serum anti-CfaE and anti-LTB antibody responses and conferred significant reduction of diarrhea compared to PBS controls (PE = 84.1%, P<0.02). These data support the further evaluation of dsc14CfaE-sCTA2/LTB as an ETEC vaccine in humans.