This tag-less CFA/I/II/IV MEFA was constructed by: 1) in silico predicting B-cell epitopes of the major structural subunits of the seven most important ETEC adhesins (CFA/I, CS1–CS6), 2) selecting one subunit (CFA/I subunit CfaB in this study) as the backbone at the criteria that this backbone subunit is very stable and relatively small-sized but carries multiple discontinuous and well-separated epitopes, 3) substituting the less antigenic epitopes of CfaB backbone with the most antigenic epitopes of the heterogeneous CS1–CS6 major structural subunits, and 4) computational modeling to optimize epitope substitution for a stable-structured MEFA. Computational modeling from the current study indicated all seven representing epitopes in the tag-less CFA/I/II/IV MEFA were surface exposed and presented at the β-sheet or the extension coil. Molecular dynamics simulation observed a low level of dynamics for these epitopes in the MEFA immunogen, suggesting these epitopes were stably presented by this MEFA protein. That correlated to the robust immune responses to each adhesin in the mice immunized with this CFA/I/II/IV MEFA.