Enteroaggregative Escherichia coli (EAEC) is an important cause of acute and chronic diarrhea worldwide. It has been shown that flagellin (FliC), a major bacterial surface protein of EAEC, causes IL-8 release from certain epithelial cell lines via activation of TLR-5. Based on the ability of this protein to activate innate immunity, flagellin can be considered as a potent adjuvant in new vaccines and adjuvant effects of native or recombinant forms of flagellin have been demonstrated. In the current study we designed various truncated forms of FliC-EAEC based on its interaction site with TLR-5 and assessed the interactions via docking protocols. Then, the most appropriate truncated forms were PCR amplified, cloned in to pGEX-5X-1 plasmid and expressed. Finally, the expressed proteins were tested for pro-inflammatory properties. Our in silico and in vitro results indicated that two truncated forms of FliC- EAEC (amino acids 79–117 and 477–508) effectively interact with TLR-5, thus could be capable of inducing in vivo immune responses.