Post-weaning diarrhea (PWD) in pigs is a continuing problem for the swine industry. The goal of this study was to utilize a concept known as reverse vaccinology towards the identification of novel vaccine candidates to be used towards the future development of a vaccine providing heterologous protection against enterotoxigenic E. coli causing (ETEC)-caused PWD in swine. The objectives used here to achieve this goal included genomic comparisons of K88+ and F18+ E. coli strains implicated in PWD, identification of vaccine candidates using a reverse genetics approach, and preliminary examination of a subset of proteins with the greatest antigenic potential. We used this approach to comprehensively analyze the porcine E. coli genomes and identify more than 50 antigenic candidates and defining loci that differentiate porcine ETEC from non-pathogenic E. coli strains. We then screened nearly 300 pathogenic and non-pathogenic E. coli from commercial pigs for the presence of these genes to identify the most discriminatory set of antigens/genes. Finally, we cloned and expressed eight of these genes to demonstrate feasibility in the development of a subunit vaccine that broadly targets porcine ETEC. This promising antigen subset represents proteins that will be further characterized and assessed for their ability to elicit a mucosal immune response in weaned pigs. The incorporation of multiple antigens into a recombinant attenuated Salmonella strain will ultimately enable heterologous protection against PWD-associated E. coli in a cost effective and reasonable manner for the swine industry.