Enterotoxigenic Escherichia coli (ETEC) are the leading bacterial cause of diarrhea.  Currently, no vaccines are available to protect against ETEC diarrhea.  ETEC strains expressing K88ac or F18 fimbriae and heat-labile (LT) and heat-stable (ST) toxins are the predominant cause of diarrhea, particularly post-weaning diarrhea (PWD), in pigs.  In this study, we genetically fused a K88ac major subunit FaeG peptide, a F18 minor subunit FedF peptide, and the LT_A2 peptide and LT_B subunit peptide of LT toxoid LT_R192G for a tripartite fusion to develop a subunit vaccine and a live vaccine strain.  Mice and piglets immunized with ‘K88ac-FaeG:F18-FedF:LT_192A2-B’ fusion developed anti-K88ac, anti-F18 and anti-LT antibodies (IgG, seIgA) which inhibited adherence of K88ac and F18 fimbrial ETEC strains and neutralized LT toxin.  Moreover, immunized piglets were protected when challenged with a diarrheal ETEC strain (K88ac/LT/STb).  The live strain expressing this fusion in a holotoxin-like structure was used to orally immunize piglets.  All immunized piglets remained healthy when challenged with the K88ac/LT/STb ETEC strain, whereas the control developed severe diarrhea and dies within 48 h.  These studies clearly demonstrated that this tripartite fusion elicited protective antibodies, and suggest this fusion is suitable for developing broadly protective vaccines against ETEC diarrhea.